Bo Liu

PhD Thesis
Biomolecular Ions in Accelerators

and Storage Rings
Bo Liu
Department of physics and Astronomy

University of Aarhus
2004
Contents
Preface i
List of Publication ii
1 Introduction
1.1 Building biomolecules 1
1.2 Soft ionization techniques 5
1.3 Study of biomolecules in the gas
phase 9
2 Experimental details
2.1 Accelerator mass spectrometer 10
2.2 ELISA 12
2.3 Sample preparation 16
3 Biomolecular ions in an accelerator mass

spectrometer
3.1 Cross section for formation of
fragment ions after collision 17
3.2 Multiply-charged oligonucleotide
anions in collisions with noble gas atoms 19
3.3 Electron attachment to nucleotide
monoanions in collisions with Na: Formation
of dianions 27
3.4 Electron capture to peptide cations 37
4 Biomolecular ions in ELISA

4.1 Decay of isolated biomolecular ions 50
4.2 Power law decay of collisionally
excited amino acids and oligonucleotides and
radiative cooling 57
4.3 Photodissociation of protonated
nucleotides and peptides 66
4.4 Lifetime measurement of radical ions 78
5 Summary and outlook 81
6 Appendix 83
7 Reference 91
Preface
The physics and chemistry of biomolecules in vacuo is a field that has
attracted considerable interest in recent years. Investigations involve both gas-
phase experiments and calculations in order to elucidate the intrinsic properties
of biomolecules under well-defined conditions and at the single-molecule level. It
is possible to vary the environment in a controlled manner, from a molecule
isolated in vacuo to a gradual build-up of a solvation shell around the molecule,
and this may lead to a better understanding of solvent effects and of the
importance of the natural biological environment.
This thesis describes the studies on biomolecular ions in the gas phase in
our lab by two apparatus: accelerator mass spectrometer and electrostatic
storage ring. High-energy collision-induced-dissociation technique is used in the
experiments of accelerator mass spectrometer and electrostatic storage ring is
used for measuring decay lifetimes of stored ions after excitation.
The thesis is organized as follows:
• Chapter 1
General introduction to biomolecules and soft ionization techniques related to my
PhD works.
• Chapter 2
This chapter deals with the experimental equipments and techniques which are
applied in the thesis. Especially the electrospray ion source is described and the
accelerator mass spectrometer and the storage ring are presented.
• Chapter 3
This chapter deals with the experiments done by the accelerator mass
spectrometer. Especially the results from the collisional experiments between
biomolecular ions and noble gas/sodium gas are presented. Ion radicals can be
generated by high-energy collisions
• Chapter 4
This chapter is devoted to the experiments done by the storage ring. Especially
the different decay laws are observed. Arrhenius parameters can be obtained by
photodissociation experiments. Lifetime of ion radicals produced by high-energy
collisions can be measured.
• Chapter 5
This is the summary of the whole thesis and the future plans are presented.
i
List of publications
1. Hvelplund P, Liu B, Nielsen SB, Tomita S, Electron capture induced dissociation
of peptide dications, Int J. Mass Spectrom. 225, 83, 2003.
2. Hvelplund P, Liu B, Nielsen SB, Tomita S, Electron capture and loss by
protonated peptides and proteins in collisions with C60 and Na, Eur. Phys. J. D 22,
75, 2003.
3. Nielsen SB, Andersen JU, Forster JS, Hvelplund P, Liu B, Pedersen UV, Tomita
S, Photodestruction of adenosine 5 '-monophosphate (AMP) nucleotide ions in
vacuo: Statistical versus nonstatistical processes, Phys. Rev. Lett. 91, 048302, 2003.
4. Andersen JU, Bonderup E, Hansen K, Hvelplund P, Liu B, Pedersen UV, Tomita
S, Temperature concepts for small, isolated systems; 1/t decay and radiative cooling,
Eur. Phys. J. D 24, 191, 2003.
5. Nielsen AB, Hvelplund P, Liu B, Nielsen SB, Tomita S, Coulomb explosion upon
electron attachment to a four-coordinate monoanionic metal complex, J. Am. Chem.
Soc. 125, 9592, 2003.
6. Andersen JU, Cederquist H, Forster JS, Huber BA, Hvelplund P, Jensen J, Liu B,
Manil B, Maunoury L, Nielsen SB, Pedersen UV, Schmidt HT, Tomita S, Zettergren
H, Power-law decay of collisionally excited amino acids and quenching by radiative
cooling, Eur. Phys. J. D 25, 139, 2003.
7. Liu B, Hvelplund P, Nielsen SB, Tomita S, Electron loss and dissociation in high
energy collisions between multiply charged oligonucleotide anions and noble gases,
Int. J. Mass Spectrom. 230, 19, 2003.
8. Liu B, Tomita S, Rangama J, Hvelplund P, Nielsen SB, Electron attachment to
"naked" and microsolvated nucleotide anions: Detection of long-lived dianions,
ChemPhysChem 4, 1341, 2003.
9. Liu B, Hvelplund P, Nielsen SB and Tomita S., Formation of C602- dianions in
collisions between C60- and Na atoms, Phys. Rev. Lett. 92, 168301, 2004.
10. Nielsen SB, Andersen JU, Hvelplund P, Liu B and Tomita S, Biomolecular ions in
accelerators and storage rings, J. Phys. B. 37, R25, 2004.
11. Andersen JU, Cederquist H, Forster JS, Huber BA, Hvelplund P, Jensen J, Liu B,
Manil B, Maunoury L, Nielsen SB, Pedersen UV, Rangama J, Schmidt HT, Tomita S
and Zettergren H, Photodissociation of protonated amino acids and peptides in an
ion storage ring. Determination of Arrhenius parameters in the high-temperature limit,
Phys.Chem.Chem.Phys 6, 2676, 2004.
ii
12. Liu B, Hvelplund P, Nielsen SB and Tomita S, Modification of Isolated DNA and
RNA Nucleotides upon Electron Attachment: Hydrogen Loss from Nucleobase
Nitrogens, J. Chem. Phys. 121, 1st Sep. 2004.
13. Tomita S, Andersen JU, Echt O, Hansen K, Hvelplund P, Liu B, Nielsen SB, and
Rangama J, Jahn-Teller splitting of the t1u and t1g orbitals in isolated C60− from near-
infrared spectroscopy, J. Am. Chem. Soc. submitted.
14. Boltalina OV, Ioffe IN, Streletskii AV, Hvelplund P, Liu B, Nielsen SB, Tomita S,
Formation of C60Fn Di-and Trianions in Collisions between fluorinated C60 and Na,
Angew. Chem. Int. ed. submitted.
15. Tomita S, Andersen JU, Cederquist H, Concina B, Huber BA, Hvelplund P,
Jensen J, Liu B, Manil B, Maunoury L, Nielsen SB, Rangama J, Schmidt HT, and
Zettergren H, Lifetimes of free C602- in a storage ring; observation of three electronic
states (in preparation).
16. Liu B, Hvelplund P, Nielsen SB and Tomita S, Photodissociation of singly
charged oligonucleotide cations (in preparation).
17. Liu B, Hvelplund P, Nielsen SB and Tomita S, Electron capture to microsolvated
AMP monoanions: Fragmentation and capture cross sections (in preparation).
iii
Introduction
_____________________________________________________________
Chapter 1
Introduction
1.1 Building biomolecules
Even though there are thousands of different molecules in a cell, there are only a few
basic classes of biomolecules. The four major classes are fatty acids, monosaccharides,
amino acids and nucleotides. These building blocks are used to build more complex and
functional biomolecules, such as proteins and nucleic acids.
Nucleotides
A nucleotide has three components: 1. a heterocyclic molecule, 2. a 5-carbon, or pentose,
sugar and 3. one or more phosphate groups. The ring-shaped molecule contains a
nitrogenous base. These bases are classified as purines (two ring-shaped molecules
joined together, one with six and one with five atoms) and pyrimidines (a single ring made
from six atoms). There are five different bases: adenine (A), guanine (G), cytosine (C),
thymine (T) and uracil (U) The first two are the larger purines while the other three are the
smaller pyrimidines (fig.1.1).
NH2 O NH2 O
N N N N N
N N N
N H2N H2N
N N N N N N
N
H H H H
Adenine Guanine Adenine Guanine
(A) (G) (A) (G)
NH2 O NH2 O
N CH3
N N H N
O
N O O O
N N N
H H H H
Cytosine
Thymine Cytosine Uracil
(C)
(T) (C) (U)
(a) (b)
Fig.1.1 Nucleobases in (a) DNA and (b) RNA.
The sugar in a nucleotide has two variants, one is called ribose and the other is
called deoxyribose. Ribose is a "normal" sugar, with one oxygen atom attached to each
carbon atom while deoxyribose is a modified sugar, lacking one oxygen atom (so the
name "deoxy"). Fig. 1.2 shows the difference between ribose and deoxyribose where in
ribose 2’ carbon is attached to OH group while in deoxyribose only hydrogen is attached to
1
Building biomolecules
_____________________________________________________________
2’ carbon. A sugar and a nucleobase can be combined by a glycosidic linkage to form a

nucleoside (fig.1.3). The molecule shown in fig.1.3 is called adenosine. When a nucleoside
joins together with a phosphate group by a phosphodiester bond, a nucleotide is formed
(fig.1.4). The molecule shown in fig.1.4 is called Adenosine MonoPhosphate (AMP).
NH2
OH OH N
N
CH2 O OH CH2 O OH
H2 N N
HO C
O
OH OH OH H
ribose deoxyribose
OH OH
Fig.1.2 Ribose and deoxyribose.
Fig.1.3 Adenosine.
NH2
7 6
N 5
N1
O 8
5' 2
N 4
N
HO P O CH2 9
O 3
OH 4' 1'
3' 2'
OH OH
Fig.1.4 Adenosine 5’-monophosphate (AMP).
Amino acids
An amino acid has three other groups attached to the α-carbon in addition to one
hydrogen: 1. an amino group (NH2), 2. a carboxyl group (COOH) and 3. a characteristic
side chain denoted as the R group. Because of this center carbon, amino acids have two
isomers due to the different steric structures, L-amino acids and D-amino acids, analogous
to left-handed and right-handed conformations. There are 20 common amino acids in a
human body and they differ only in the side chain group.
H
H H2N C COOH
H2N C COOH H2N C COOH
H2 CH2
CH2
H CH2
Glycine C
CH CH2
HC CH CH2
N C
H H NH2
Tryptophan Lysine
Fig.1.5 Amino acids with typical side chain groups.
2
Introduction
_____________________________________________________________
The simplest amino acid is glycine whose side chain is only a hydrogen atom
(fig.1.5). Some side chains contain an aromatic ring such as tryptophan that enables them
to absorb UV light and some side chain has another amino group that is easily protonated.
In fig.1.5 the amino acids with typical side chain groups are shown.
NH2
N NH2
N
H
H N
N
O N 5' H
N
HO P OCH2 O H
O N N
OH HO P OCH2 O
OH NH2
OH H + H2O
NH2 N
H N
H
+ N
N O N
H
H N
H O P OCH2 O
N N
OH
OH
O P OCH2 O
OH OH H
OH H (a) 3'
R1 R2 R1 R2
O O O O
H2N CH C OH + H 2N CH C OH H2N CH C N
H
CH C OH + H 2O
(b)
Fig.1.6 Polymerization of (a) nucleotides and (b) amino acids.
From brick to mansion

Nucleic acids and proteins are gigantic molecules, for example, if all the DNA
(DeoxyriboNucleic Acid) molecules in a human’s body are connected with each other, one
has about 9 million kilometers of DNA. That is enough to reach to the moon and back 13
times. Such big mansions need millions of small bricks. As mentioned above, the building
blocks for nucleic acids are nucleotides while those for proteins are amino acids. The
process which stitches the single nucleotide or amino acid (monomer) together is called
polymerization. In fig.1.6 the polymerization process for nucleotides and amino acids is
shown. Polymerization is a dehydration condensation process which takes one –OH from
one monomer and one hydrogen from a NH or an OH group of the other monomer to form
water. The bond to link monomers in nucleotide is a phosphodiester bond and in the case
of peptides it is a peptide bond. A few monomers can be knitted to form an oligomer, for
instance, an oligonucleotide is made from nucleotides and a peptide is made from amino
acids. Specific atoms in a molecule are identified by numbering the backbone atoms: C1,
3
Building biomolecules
_____________________________________________________________
C2, etc. In a complex nucleotide, the atoms of the purine or pyrimidine ring are first
numbered 1, 2, 3, etc. Carbon atoms in the sugar are then numbered 1', 2', 3', 4', and 5'
(see fig.1.4). A nucleotide has two ends: One end of the chain carries a free phosphate
group attached to the 5'-carbon atom; this is called the 5' end of the molecule. The other
end has a free hydroxyl (-OH) group at the 3'-carbon and is called the 3' end of the
molecule (see fig.1.6). Also a peptide has two terminals: one end of the chain has the
amino group called the N terminal; the other end has the carboxyl group called the C
terminal.
(a) (b)
Fig.1.7 (a) DNA double helix (b) RNA hairpin loop.
DNA is the body's genetic architectural blueprint present in almost every cell of the
body. In 1953 the double helix structure was discovered by Watson and Crick who
deduced the base pairing between the two DNA molecules from the double helix structure
(fig.1.7a) [1,2]. It is the sequence of DNA that stores the inherited information. Ribonucleic
acid (RNA) molecules are usually single-stranded but by formation of hairpin loops RNA
can also form a double-helical structure (fig.1.7b) [3]. Because of the influence of the steric
interaction and hydrogen bonds, Watson and Crick deduced that adenine must pair with
thymine and guanine must pair with cytosine. In hairpin loop RNA, adenine pairs with
uracil since in RNA, thymine is replaced by uracil (fig.1.8).
4
Introduction
_____________________________________________________________
H3C O H NH N H H HN H O N H
H N H N N
H N H N
N N N N
O H O H NH
Thymine Adenine Cytosine Guanine
Fig.1.8 Watson-Crick base paring.
Proteins are the most abundant and diverse molecules found in living cells. They
typically make up about half the total weight of biomolecules in a cell (excluding water).
Proteins can be enzymes, specific carriers as hemoglobins that carry oxygen (fig.1.9),
and are important in contraction, such as actin fibers that interact in muscle tissue, etc.
Fig.1.9 Hemoglobin.
1.2 Soft ionization techniques

Biomolecules are studied intensively in solution and in the solid state. The structure of
molecules can be elucidated using x-ray crystallography. It is however difficult to study
biomolecules in the gas phase simply because the biomolecules are so fragile (the
weakest covalent bond dissociation energy is of the order of 1 eV). Before the late 1960's,
the ionization techniques available, such as electron impact ionization or photoionization
could not be used for ionizing large biomolecules. The reason is that biomolecules are
heated up during the ionization process and thus the biomolecules therefore decompose.
A "gentle" ionization technique was therefore required. During the 70's chemical ionization
(CI) [4], plasma desorption (PD) [5] and fast atom bombardment (FAB) were invented [6],
enabling scientists to produce large biomolecular ions. These techniques achieved certain
success, however, for biomolecules larger than 10 kamu it was difficult to ionize them in
the gas phase, which meant that these techniques were not "soft" enough. In the late 80's,
with the invention of true "soft" ionization techniques such as electrospray ionization (ESI)
[7,8] and soft laser desorption ionization (SLD) [9,10], it became possible to generate
5
Soft ionization techniques
_____________________________________________________________
even heavier biomolecular ions such as proteins and nucleotides of 20 kamu in the gas
phase. The ionization technique applied in my work is ESI, so I will discuss some details
about ESI.
Fig.1.10 Formation of Taylor cone at the needle.
Electrospray ionization
The idea behind ESI was first proposed by Dole in 1968 [11]. Fenn developed Dole’s idea
and in 1984 he successfully coupled ESI to a mass spectrometer (MS) and in 1988 he
measured biomolecules of 40 kamu. Briefly, there are three major steps to form the gas-
phase ions in ESI: (1) formation of charged droplets in the tip of a capillary, (2) shrinkage
of the droplets into small highly-charged droplets, and (3) formation of gas-phase ions
from small highly-charged droplets.
Coulomb fission
Taylor jet-cone
Fig.1.11 Scheme of formation of small charged droplets.
In ESI, the needle that contains dissolved biomolecules is put to a high electric
voltage. When the electric field is strong enough to penetrate the liquid, it leads to the
formation of a jet cone and droplets are emitted (fig.1.10) [12]. Smith [13] deduced a
useful equation for the required electric field at the capillary tip, which shows that the
onset voltage is proportional to the square root of the surface tension of the solution.
6
Introduction
_____________________________________________________________
Therefore, a solution that has a high surface tension is difficult to spray. For example, in
Smith’s paper, they needed 4 kV to spray a water solution while a voltage of 2.2 kV is
enough to spray a methanol solution. As the solvent molecules evaporate from the
droplets, the droplets shrink. The density of charge increases which means the Coulomb
repulsion also increases. When the Coulomb repulsion is equal to the surface tension of
the droplet, fission occurs and the droplet divides into several small droplets. Figure 1.11
is the scheme of the first two steps of ESI.
m/q
Fig.1.12 Electrospray ion mass (m/q) spectra

measured for disulfide-bond reduced lysozyme.
There are however two theories for the formation of gas-phase ions from the small
highly charged droplets. Dole and Röllgen proposed a charged residue method in which
the fission would continue until only one excess ion is left [11,14]. Iribarne and Thomson
proposed a different mechanism, called the ion evaporation method [15,16], in which the
radius of a droplet decreases until direct ion emission is possible. When the radius is less
than 10 nm, ion emission dominates over fission. Experiments have been done to
demonstrate the validity of the two theories and the results show that the ion evaporation
model is preferred to the charged residue model [8,17,18], but there has been no decisive
experiment to distinguish the two mechanisms.
The charge state distribution of the final ions depends on the solution pH [19-21],
solvent [22,23], analyte concentration [24,25] and counterion [13,25] in solution, etc. for
example, variation of solution pH can change the acid-base equilibrium thus altering the
degree of positive or negative charging through protonation or deprotonation. With a
higher polarity of the solvent molecules, a higher degree of charge separation can be
achieved leading to ions with a higher charge state. In general, electrospray tends to
generate multiply charged ions, especially for molecules with molecular weight larger than
2 kamu. This allows analysis of high molecular weight proteins using analyzers with a
lower mass/charge limit. In Fig.1.12 a typical ESI spectrum of the protein lysozyme
(15154 amu) is shown [26].
Soft Laser desorption

In 1987 Tanaka presented results for intact proteins in the gas phase with the
weight more than 10 kamu. He pointed out that the key to ionize large biomolecules was
the proper laser energy and wavelength with high absorbance and a good matrix to
transfer heat. Karas and Hillenkamp developed a new crystalline matrix with an
absorption maximum matched to the wavelength of the laser pulse. The technique is
7
Soft ionization techniques
_____________________________________________________________
called matrix-assisted laser-desorption ionization (MALDI) [27,28]. Nowadays, the

predominant SLD technique is MALDI.
Fig.1.13 Scheme of process of MALDI.
In principle, there are two major steps to form gas-phase ions in SLD: (1) after
absorbing the photon energy emitted by a laser, matrix molecules together with the analyte
molecules are sublimated into the gas, and (2) the irradiation of the matrix induces
electronic excitation of the matrix molecules, accumulating large amount of energy. Gas-
phase ions are formed through proton transfer between the matrix molecules and the
analyte molecules. Figure 1.13 shows the principle of SLD in the form of MALDI. One of the
properties of SLD is that the resulting ions are dominantly singly charged.
Fig.1.14 Sonic spray ionization source from reference [31].
8
Study of biomolecules in the gas phase
_____________________________________________________________
Sonic spray ionization

Sonic Spray Ionization (SSI) was developed in the 1990’s by Hirabayashi [29,30]. The
original idea was to couple the liquid chromatographs (LC) and capillary electrophoresis to
mass spectrometry. The property of SSI is its simplicity since no heating or high voltage is
needed. This source thus delivers ions with very low internal energy such as clusters
(fig.1.14). Cooks observed magic number of serine clusters and also studied the chirality
recognition of L- and D- amino acids [31-33].
2000
1800
1600
1400
1200
Papers
1000
800
600
400
200
0
-1991 92-93 94-95 96-97 98-99 00-01 02-03
Year
Fig.1.15 Research on biomolecules in the gas phase.
1.3 Study of biomolecules in the gas phase

After Fenn interfaced ESI to MS for the first time, intensive studies of biomolecules in the
gas phase have been made. Collision-induced dissociation (CID) experiments can
determine the sequence of peptides and nucleic acids [34,35]. It can also be used for
studies of the dissociation dynamics of molecules. Recently, a technique called electron
capture dissociation (ECD) was developed [36,37]. Compared to CID, ECD can induce a
non-ergodic process which causes special fragmentations in biomolecules. Blackbody
infrared radiative dissociation (BIRD) experiments can determine the activation energy
and its corresponding preexponential factor for a unimolecular fragmentation [38,39]. Ion
mobility experiments can reveal the conformation of a molecule depending on charge
state or environment [40,41]. Photoelectron spectroscopy (PES) can be used to obtain
the electron affinity of a molecule [42,43]. In other words, studies of biomolecules in the
gas phase using soft ionization technique are a hot research field nowadays (fig.1.15) [44].
9
Experimental details
_____________________________________________________________
Chapter 2
Experimental details:
2.1 Accelerator mass spectrometer

A tandem mass spectrometer is used in the collision experiments between biomolecular
ions and target gases (fig.2.1). This equipment has five major parts: (1) ESI source, (2)
accelerator tube, (3) magnet sector, (4) collision cell and (5) electrostatic analyzer. The
equipment is operated in the way described in the following:
gas collision cell

Magnet
− + Electrostatic
analyzer
Channeltron 50 kV
detector
ESI source
Fig.2.1 Schematic drawing of accelerator mass spectrometer.
The ESI source, together with rotary pumps and turbomolecular pumps, is mounted
on the high-voltage platform of the accelerator. A stainless steel hypodermic needle is, via
a fused silica capillary, connected to a syringe containing a solution of the analyte. A
syringe pump (Harvard apparatus) delivers a constant flow through the needle. Increasing
the flow rate helps to stabilize the signals. A typical flow rate is 1µl/min. A voltage of 3-4kV
relative to the heated capillary is put on the needle. The electrospray produces highly
charged droplets at atmospheric pressure and gaseous, multiply charged analyte ions are
formed from the droplets in the heated capillary. The capillary is a 10-cm long stainless
steel rod with a 0.4-mm bore, normally heated to 180 °C for desolvation of water from
analyte molecules. At the exit of the capillary ions emerge into the first vacuum zone
where a rotary pump maintains a pressure of around 1 Torr. The ions focused by a tube
lens go through a skimmer into a second region. The voltage on the tube lens varies from
50 V to 200 V depending on the charge state of the desired ions [45,46]. This voltage
dependence is due to in-source CID between biomolecules and residual gas. In-source
CID can also lead to molecules with higher internal energy because the ions can have
higher kinetic energy with higher tube lens voltage and collisions with gases heat up the
“cold” molecules. This effect was observed in our lab (see appendix). The pressure in the
second region is further reduced to 1 mTorr by a turbopump. The ions are steered through
this section by an octopole beam guide, and enter the third vacuum region through lenses.
10
Accelerator mass spectrometer
_____________________________________________________________
This region is evacuated to 10-5 Torr by a second turbopump. An einzel lens assembly
focuses the ions into the acceleration tube of the isotope separator in which the potential
difference is 50 kV. The schematic drawing of the ESI source is shown in fig.2.2. After
acceleration, an m/q analysis (m being the mass of the ion and q being the charge state) is
performed by a large, 2-m radius, 72° bending magnet capable of deflecting singly
charged ions with mass up to 5000 amu. After magnetic analysis the mass-selected ions
pass through a target gas contained in a 3-cm long differentially pumped cell with entrance
and exit apertures of 1 and 3-mm in diameter, respectively. Then the ions enter a
computer-controlled 180° hemispherical electrostatic analyzer (ESA) with a radius of 15
cm. Finally, the transmitted ions are detected by a channeltron connected through
amplifiers and discriminators to the PC that is operated in the particle counting mode.
Tube lens Octapole
Lenses
ESI needle
Skimmer Acceleration tube
4kV
1 mbar 10-3 mbar 10-5 mbar 10-6 mbar
Rotary Turbo Turbo

Heated
pump pump pump
Capillary
(~180°C)
Fused silica
capillary
Fig.2.2 Schematic drawing of electrospray ion source.
The principle of the ESA for mass analysis is that only ions with proper kinetic
energy can pass through. The physics behind is that the centripetal force equals the
electrostatic force that the ions experience, i.e.
U mv 2
q ESA =
d r
(1),
1 d
U ESA = × × 2E kin
q r
where v is velocity of the ions, respectively, UESA is the potential on the ESA, d is the
distance between two conducting half spheres and r is the radius of the trajectory. From
eq.1, the scan of UESA means the selection of ions with different kinetic energy per charge
state. In our case, if precursor ions fragment in the collision cell, the velocity of the
fragments is the same as that of the precursor; therefore, scanning the electric field can
separate the fragments with different m/q ratios. This method of analysis is called mass-
analyzed ion kinetic energy (MIKE) spectrometry.
11
_____________________________________________________________
2.2 ELISA
Recently, an ELectrostatic Ion Storage ring Aarhus (ELISA), figure 2.3, has been built at
the University of Aarhus [47,48]. Briefly, ELISA contains two 160° cylindrical deflectors
with four 10° parallel-plate deflectors and two electrostatic quadrupole doublets. Ions can
be stored using suitable parameters and neutrals originating from precursor ions are
detected by the channel plate at the end of one of the straight parts of ELISA. Since it is
electrostatic, ions with the same energy-to-charge ratio can be stored using fixed
parameters (mass independent). ELISA is compact (the circumference is 8.35m). The
vacuum in ELISA is 10-11 mbar, but when a geometrical cross-section measurement is
performed, the pressure in the ring is increased to increase the production of neutral
fragments. A pulsed laser can be applied on the side opposite to the detector to interact
with the stored ion bunches. If the stored ions are multicharged, the production of neutral
fragments could be very low and a channeltron detector is put just after one of the 10°
deflectors. The position of the channeltron relative to the beam trajectory depends on the
mass-to-charge ratio: if the m/q ratio of fragments is higher than that of the precursor i.e.
fragments bent less than precursor, then the detector should be placed at a smaller angle
relative to the trajectory. On the other hand, if the m/q of the fragments is lower than that of
the precursor the detector should be put at a larger angle relative to the trajectory. When
the injection beam line of ELISA is coupled to an electrospray ion source, biomolecular
ions can be studied. The spray source used in ELISA is the same as the one in the
accelerator MS instrument except that an 22-pole ion trap is used to store the beam and
make ion-bunches for injection into ELISA. The ion trap can operate in two modes, trap
mode and extraction mode. When it is in the trap mode, ions are accumulated. In the
extraction mode, ions are expelled from the trap and injected as an ion bunch into ELISA.
The ion trap is placed in the ESI source in the third stage before the Einzel lens. A voltage
of 22kV is applied to the acceleration tube to accelerate the ions. After acceleration the
ions are selected by a bending magnet according to their mass-to-charge ratio.
Channeltron detector
Ion bunch
Magnet
Na oven Microchannel
1m plate detector
Accelerator with
electrospray ion source
Fig.2.3 Schematic drawing of the electrostatic storage ring (ELISA).
Typical timing sequence for ELISA

A typical timing sequence is shown in Fig. 2.4. The electrospray ion source runs
continuously and ions are stored in the trap as shown at the top of Fig. 2.4 by the sine
wave corresponding to the RF applied to the trap. A timer starts to generate a start signal,
12
ELISA
____________________________________________________________
and after a fixed delay, a timing signal is sent to the computer to start the recording
program, and also a signal to the ion trap to extract the stored ions. This extraction mode
lasts several tens of microseconds, then the trap returns to the trap mode. After a delay of
1ms after the start signal, ELISA closes to circulate the bunched ions from the injection
beam line. Different mass-to-charge ratios determine the flight time from the ion trap to the
entrance of ELISA, determining the timing signal to the ion trap. Therefore, signals for
extracting heavier ions must be earlier than those for lighter ions. In the timing sequence of
10 Hz above, nearly 100 ms is spent in the trap mode. A voltage can be put to the skimmer
(fig. 2.2) to have different storage time, and therefore different timing signal can give
different ion storage times. A typical timing for the laser is about 50 ms after the start signal.
Because of some internal delay of the laser, the input timing for the laser is not the same as
the real timing when laser irradiates. The actual timing of the laser is an important
parameter because the laser pulse should overlap with the bunched ions. We monitor the
Q-switch timing to obtain the actual time for the laser timing. In order to achieve maximum
overlapping, we need to fine-tune the timing for the laser.
start PC
skimmer signal
extraction signal
ions accumulation
ELISA close timing

1 ms
Fig.2.4 Typical timing sequence for ELISA.
The 22-pole ion trap

The ESI source used in ELISA is almost identical to the one used in the accelerator MS
except that a 22-pole ion trap is included. An ion trap should have (1) even-numbered
electrodes that can trap the ions in the radial direction and (2) entrance-cap and end-cap
ring electrodes that confine ions in the axial direction (fig.2.5 shows a quadrupole). A DC
voltage is on the entrance-cap and end-cap ring electrode to prevent the ions from
escaping. A RF voltage, U ± V cos(Ωt ) , is applied on the multipole electrodes. Ions in such
conditions have two kinds of motions in the radial direction in the trap: (1) the motion
around the center called secular motion whose frequency is quite slow compared with the
RF frequency, Ω and (2) micromotion that has a frequency as high as the RF frequency
and the center of micromotion is the simultaneous position where the secular motion
13
_____________________________________________________________
moves. The amplitude of micromotion is proportional to the instantaneous amplitude of the

secular motion that means in the return point of a secular motion, micromotion has its
highest amplitude. In fig.2.6 is shown the combination of the two motions. From this figure,
it is clear that the frequency of the secular motion is much lower than that of the
micromotion, and the amplitude of the micromotion is at the maximum when the ions are
on a return point. The secular motion of the ions can be considered to be driven by an
effective potential (or psedopotential).
U − V cos(Ωt )
U + V cos(Ωt )
Fig.2.5 Illustration of quadrupole ion trap.
The advantage of a 22-pole ion trap is that it can create a effective potential with a
wider flat field-free region compared with that of quadrupole around the center trap. The
ions traveling in the field-free region therefore have a diminishing micromotion. Therefore
heating due to coupling from micromotion is also smaller. For example Gerlich [49]
simulated temperature distributions for the storage of C+ in a H2 buffer gas at 80 K and the
result was that a high tail was found both in multipole ion traps and quadrupoles, but in a
quadrupole the tail was much higher than that in a multipole ion trap (see [49] for more
details, see [50,51] for recent experiments based on 22-pole trap).
Fig.2.6 Combination of secular motion and micromotion.
14
ELISA
____________________________________________________________
In fig.2.7 the schematic drawing of the 22-pole ion trap in our lab is shown. The 22-
pole ion trap follows the design of Gerlich [52,53]. It has a length of about 4 cm and the 22
rods with 1-mm radius form a cylinder with a radius of 5 mm. There are electrodes at each
end to stop and extract ions. The five thin ring electrodes located just outside the 22
electrodes produce a weak electrostatic field in the axial direction for manipulation of the
ions inside the trap. The RF voltage applied to the multipole electrode has an amplitude of
100 V and a frequency of 3 MHz. A DC voltage of several volts is applied to the entrance
and end cap ring electrodes.
Fig.2.7 Illustration of 22-pole ion trap.
AMP anions were used to test the ion trap. Fig. 2.8a shows a pickup signal [47] for a
single bunch of deprotonated AMP ions circulating in ELISA. The trap ran in the mode of 1
Hz i.e., ion accumulation time is about 1 s (no voltage on skimmer). From the figure, the
revolution time of AMP anions is determined to be 75 µs that agrees with the calculated
value for this ion of mass 346 and with 22 keV kinetic energy. Fig. 2.8b shows the details
around the peak. The time required to empty the trap is about 40 µs that should be less
than the width of the extraction signal, 50 µs. Moreover, the peak area is proportional to
the number of the ions in the bunch. Table 2.1 gives the details about the peak area and
the corresponding number of ions. Comparing the number of ions with the storage times of
1 s 10 s and 25 s, it is found that the number of ions saturates. The reason for this is the
space-charge effect that destroys the stability status of a trap leading to loss of ions. So
the number of ions in the 10 s storage is only a factor of 5 higher than that in the 1 s
storage.
Table 1 number of ions in a bunch (calibration based on [54]).

Repetition time Peak area Number of ions
(mV? µs) (? 103)
1 Hz 5.65 226
0.1 Hz 26.1 1044
0.05 Hz 21.9 876
15
_____________________________________________________________
1.0
(a) 75 µs
0.8
Σ (mV) 0.6
0.4
0.2
0.0
0 200 400 600 800 1000
Time (µs)
1.0
(b)
0.8
0.6
Σ (mV)
0.4
0.2
40 µs
0.0
400 420 440 460 480 500
Time (µs)
Fig.2.8 (a) Pickup signals for AMP anions stored in

the 22-pole trap (b) the details of one peak.
2.3 Sample preparation

Mononucleotides and peptides were brought from SIGMA-Aldrich and oligonucleotides
were from DNA Technology, Denmark. The solution used for spraying anions was formed
by diluting to yield 20 µM in a methanol/H2O (1:1 volume) solution. All the solutions for
cations are formed by diluting to yield 20 µM in an acetic acid (0.1%) water/methanol (1:1)
solution. The flow rate is set to 2 µl/min for the “anion solution” and 1 µl/min for the “cation
solution”.
16
Biomolecular ions in an accelerator mass spectrometer
_____________________________________________________________
Chapter 3
Biomolecular ions in an accelerator mass

spectrometer
3.1 Cross section for formation of fragment ions after
collision
After collision with the target gas, both fragment ions and parent ions are collected by an
ESA, which enables one to measure the production cross section of a fragment ion by the
so called growth rate method [55]. An example is given below to demonstrate this method.
If an anion beam with a high translational energy is passing through a gas target, an
extra electron can be captured from the target gas to the parent ions (fig. 3.1). Assuming
that the density of target gas is 1, at any position x, the intensity changes of the primary
ions is
dI −
= −σ t − I − (1)
dx
where σ t − is the destruction cross section for the primary ions. At the position x, the
intensity changes of the dianion is
dI 2 −
= σ eI − − σ t 2− I 2− (2)
dx
where σ e is the electron capture cross section and σ t 2 − is the destruction cross section for
dianions.
From eq. (1), the intensity of primary ions can be found as
−σ − x
I − = I 0− e t
(3)
−
where I is the intensity of primary ions before passing through the target.
0
Substituting (3) into (2), we have

dI 2 − −σ − x
+ σ t 2 − I 2 − = σ e I 0− e t (4)
dx
This is a linear non-homogenous differential equation, and its analytical solution is
−σ x σ e I 0− (σ −σ )x σ e I 0−
I 2 − (x) = e t 2 − ( e t2− t− − ) (5)
σ t2− − σ t − σ t2− − σ t −
However, what we can measure in the spectrum is the intensity of dianions and primary
−σ x
anions passing through the target gas, i.e., I 2 − (x) and I − (x) which is equal to I 0− e t−
. So
eq. (5) is revised to
−σ − x (σ − −σ 2 − )x σ e I 0− (σ 2 − −σ − )x σ e I 0−
I 2 − (x) = e t ⋅ e t t ( e t t − ) (6)
σ t2− − σ t − σ t2− − σ t −
Finally, we have
−σ − x σ e I 0− σ e I 0− (σ − − σ 2 − )x
I 2 − (x) = e t
⋅( − e t t ) (7)
σ t2− − σ t − σ t2− − σ t −
17
Cross section for formation of fragment ions after collisions
_____________________________________________________________
So if the destruction cross section between anion and dianion is small, i.e., (σ t − − σ t 2 − )x is
a small value, we can expand the exponential, and eq. (7) becomes
1
I 2 − (x) = σ e I − (x)(x − (σ t 2 − − σ t − )x 2 ) (8)
2
From eq.(8), if we neglect second order effects, the ratio between the intensity of dianions
and primary anions after collision is proportional to the target thickness. Deviations from a
linear increase then give information about the difference between the destruction cross
sections for these two ions. An example will be shown in section 3.2.
The reason why the electron can be transferred from one ion to another can be
explained by the classical over-the-barrier model. In the mid-1980s, this model was
developed for charge transfer in slow ion-atom collisions [56-58]. This model is useful for
estimates of the total and absolute cross sections for electron removal from an atomic
target. The atomic models assume that electron transfer is possible when the over-the-
barrier condition is fulfilled, i.e., when the potential seen by the electron moving from the
target to the projectile equals the Stark-shifted binding energy for the active electron at the
target. For big molecules such as biomolecules or clusters, the point charge approximation
is no longer valid, so more sophisticated treatments of the targets and projectiles are
needed. Recently, Cederquist and coworkers [59, 60] applied the over-the-barrier model to
treat the electron transfer process in collisions between C60/Arq+ and multiply charged
C60/C60 ions. They considered the C60 ion as a conducting sphere and the Arq+, for
instance, as a point charge. In this case, the potential outside of the conducting sphere
can be calculated by means of electrostatic image charges (see Fig.3 in ref. [59]).
I 0− I − (x) I − (l)
I 2 − (x) I 2 − (l)
0 x
Fig.3.1 Schematic drawing of the collision process. The intensity of the

primary beam is I 0− and when the beam arrives at point x, the intensity
of the primary beam and the dianion are I − (x) and I 2 − (x) , respectively.
When they leave the collision cell, the intensities of these two ions
measured by ESA are I − (l) and I 2 − (l) .
18
_____________________________________________________________
3.2 Multiply-charged oligonucleotide anions in

collisions with noble gas atoms
Total destruction cross sections
The total destruction cross section was measured for [d(A)7-nH]n- in He, Ne and Ar by the
beam attenuation technique for n between 2 to 5. The cross sections are shown in Fig. 3.2
and it should be noted that Ne and Ar give almost identical values whereas the cross
sections in He are around 15% lower and the cross sections increase slowly with projectile
charge state. We have earlier observed similar behaviors for high energy collisions
between lysozyme ions and noble gases [61] and will adapt the model used then in the
present case. The d(A)7 molecule (formula: C70H79N35O33P6) is approximated by a sphere
with a radius of 6.8 Å based on the total destruction cross section in collisions with Ar or
Ne. The destruction cross section for collisions with He has to be modified since the
molecule is transparent to He atoms. The cross section for transfer of more than around 5
eV (a typical binding energy) to an atom in the molecule in a collision with a He atom is σ =
1.1 Å2. The average projected density of heavy atoms in the d(A)7 molecule is around t=1
atom/Å2 and we estimate the transparency factor by its value for a random two-
dimensional distribution, T=e-tσ which gives T=0.32 for He and a negligible value for Ne
and Ar. Multiplying the geometrical cross section by (1-T) we then arrive at a cross section
of 120 Å2 for a He target as compared to around 200 Å2 for Ne and Ar targets. We believe
that this simple model, in a qualitative way, explains the difference in cross sections
measured in light and heavy target gases, respectively. From Fig. 3.2 it is evident that the
measured cross sections increase as a function of projectile charge state. A similar charge
state dependence of cross sections measured by ion mobility was reported earlier by
Hoaglund et al. [62] for charge states between 2 and 6 of 10-residue oligonucleotide
anions comprised entirely of thymine bases. They ascribed the increase in cross section
with charge state to an unfolding of the molecule as a result of Coulomb repulsion. We
believe that the increase in total destruction cross section as a function of charge state
which we observe can also be explained by unfolding due to Coulomb repulsion.
250
He
200 Ne
Ar
Cross section (Å )
2
150
100
50
0
0 1 2 3 4 5 6
n
Fig.3.2 Total destruction cross sections for [d(A)7-nH]n- in He, Ne or Ar gas as a function of n.
19
Oligonucleotide anions in collisions with noble gas atoms
_____________________________________________________________
2−
[d(A ) 5 − 2H ]
−
[d(A ) 5 − 2H ]
3−
[d(A ) 5 − 3H ]
2−
[d(A ) 5 − 3H ]
−
[d(A ) 5 − 3H ]
0 200 400 600 800 1000 1200 1400 1600
m /q
Fig.3.3 Spectra obtained for collisions between doubly and triply
deprotonated d(A)5 and He. Note that the dominating “fragment” peaks
correspond to single and double electron loss.
Single and double electron loss from oligonucleotides

When multiply-charged anions collide with noble gas, one or two electrons can be lost
from the precursor anions (fig. 3.3). From fig. 3.3, it is seen that electron loss from
precursor ions occurs and that electron lost radical ions dominate over the other fragment
ions. This is also observed in peptides and proteins in high energy CID [63, 64], but is not
observed in low energy CID since high energy is required in order to satisfy the Massey
criterion [65] i.e., when the electron transfer time is equal to the collision time, the
maximum electron capture cross section can be obtained. In fig. 3.4 is shown the ratio of
the intensity of one electron reduced [d(A)5-3H]2- and its precursor [d(A)5-3H]3- as a
function of He target thickness. The electron capture cross section can be obtained by a
linear fit. The slope gives the cross section as discussed in the section above. In this way
cross sections for single or double electron loss can be obtained as a function of charge
state in different target gases. In fig. 3.5 the single electron loss cross section of dA7 in
three gases is shown. From this figure, it can be seen that the capture cross section is
proportional to the charge state, i.e. to the number of charged phosphate groups. The
single electron loss cross sections of dA73- and dA53-, whose sizes are different, have
similar values (19.2 and 18.5 Å2, respectively). This means to a first approximation that the
only factor that can influence the electron loss cross section is the number of charged
phosphates, and the other factors, such as the size of the precursor ions can be neglected.
20
_____________________________________________________________
It should also be stressed that we have never observed any sign of three electron loss in
the collision systems investigated. The ratio between double and single electron loss was
found to be insensitive to both projectile charge state and target gas and amounted to
around 0.25. The observations listed above allow us to propose a simple model for
electron loss from nucleotide anions where the number of negatively charged phosphate
groups is the decisive parameter. We assume that a number of separated O− ions
attached to individual phosphate groups are responsible for the final charge state of the
nucleotide. It is thus tempting to model the nucleotide anion as an assembly of
independent oxygen anions imbedded in a passive medium. By linear fitting, the single-
electron loss cross section (σe) of an individual oxygen anion could be obtained from the
slope of the line. In Table 3.1, a comparison is shown between our experimental data
obtained as described above and previous experimental data obtained for O− ions [66, 67].
This comparison is rather crude since the data for the atomic ions are based on
reasonable extrapolations of the original measurements. On the other hand the favorable
comparison between the two sets of data indicates that our simple model contains the
essentials of the interaction between oligonucleotide anions and rare gas atoms in keV
collisions.
0.08
3−
[dA 5− 3H] +He
0.07
0.06
Relative intensity
0.05
2
σ=18.5Å
0.04
0.03
0.02
0.01
0.00
0.0 1.0x10
-3
2.0x10
-3
3.0x10
-3
4.0x10
-3
2
Target thickness (/Å )
Fig.3.4 Single electron loss cross section for [dA5-3H] 3- in He.
The observation that all the cross sections for double-electron loss amount to 25%
of the cross sections for single-electron loss indicates that the two electrons are lost from
the same O− ion in the molecule. A rough estimate shows that if two different O− ions were
responsible for double-electron loss then the cross section should at most amount to a few
percent of the cross section for single-electron loss and also depend on the charge state.
Therefore, the double-electron loss process is most likely caused by autoionizing states in
the phosphate group after one electron is lost from O−.
The biomolecules are transparent to He atoms as described in the section about total
destruction cross sections. This transparency will also influence the electron loss cross
section. A He atom may penetrate the molecule and cause electron loss without
fragmentation whereas a larger atom like Ne or Ar can cause only pure electron loss in
21
_____________________________________________________________
glancing collisions. This effect could explain why the measured electron loss cross section
is around 40% larger for He than for Ne and Ar. We thus conclude that He is the ideal
target gas if we aim at having large electron loss probabilities and small collisional induced
fragmentation probabilities at the same time. Comparing Ar and He can tell us whether O-
is located on the surface or in the interior.
40
electron loss cross section of dA7
30
Cross section (Å )
2
20
He
10
Ar
Ne
0
0 1 2 3 4 5 6
Charge state
Fig.3.5 Electron loss cross section of dA7 as a function of charge state.
Table 3.1 Comparison of the single electron loss cross section of oxygen anions deduced
from our experiments and the results from Matić and Čobić [66] and Penent et al. [67].
target gas σe (our exp.)/Å2 σe (O- exp.)/Å2
He 6.5 4.5 [67]
Ne 3.2 2.0 [66]
Ar 3.8 3.5 [66]
Electron-loss induced dissociation

The MIKE spectrum for two 5-mer oligonucleotides [d(A)5-2H]2- and [d(GCCCC)-2H]2- is
shown in fig. 3.6. in addition to the electron loss peak, there are many fragment ions. These
ions come from collisionally activated (induced) dissociation (CAD or CID). The CID
process typically has two steps: (1) a fast collision process (10-14 to 10-16s) when a fraction
of the ion translational energy is converted into the ion internal energy bringing the ion to an
excited state, and (2) the energy is redistributed within the internal vibration modes, and
unimolecular decomposition occurs. Depending on the collision energy, the CID technique
can be divided into high-energy CID and low-energy CID. There is no rigorous definition
about high-energy CID and low-energy CID. Roughly the typical energy of high energy CID
is several keV for 1000 amu ions. In low energy CID, the typical energy ranges from 1-100
eV. Both high- and low-energy CID techniques have been extensively used in sequencing
22
_____________________________________________________________
of peptides and nucleotides. Because of the difference in the energy transferred, the
fragmentation spectrum is quite different. The peaks in a low energy CID spectrum are few
because the bond cleaved is that of low dissociation energy. In the high energy CID
spectrum, more fragmentation channels are open so that the number of peaks is enhanced.
Comparing the spectrum taken in low energy CID with that in high energy CID, one can
have extra information about the molecules.
EC
a)
w4
2−
[−A]
w3a4−B4 [−A]
−
a2−B2
w2 ∑4
a4
w1 a3 ∑3
0 200 400 600 800 1000 1200 1400 1600

EC
b)
w4
a4−B4 Σ4
w3
w1 a −B Σ3 d4 [−C]−
2 2 w d3
a2 2 a4
a3
0 200 400 600 800 1000 1200 1400
m/q
Fig.3.6 CID spectra of two different 5-mer dianion oligonucleotides in
collisions with He gas (a) [d(A)5-2H]2- and (b) [d(GCCCC)-2H]2-.
In the beginning of the 90’s, low-energy CID experiments on oligonucleotide anions

were preformed by McLuckey [68-70], and he pointed out that in low energy CID, there
are two steps involved in the fragmentation: (1) loss of a nucleobase as a neutral or
charged base, and (2) cleavage of the 3’C-O bond of the sugar from which the base was
lost to produce the complementary “w” and “a-B” type ions (scheme 3.1) using his
nomenclature for the fragments. These two types of fragments are useful in sequencing,
for instance, the mass of B3=w3-w2-mass (sugar + phosphate). More sequencing of
oligonucleotides was made for negative ions since it is easy to remove a proton from
phosphoric acid. Very recently, progress has been made in sequencing oligonucleotides
for cations [71-73]. The fragmentation pathway is similar for positive ions to that for
negative ions, i.e., loss of a positively charged base followed by 3’ C-O bond cleavage
23
_____________________________________________________________
leading to w and a-B ions. The charged site is the nucleobase instead of the phosphate in
negative ions, so the abundance of the fragment ion, a-B, depends on the possibility of
base protonation; for example, thymine has a low proton affinity, therefore losing a
charged thymine base is unfavorable, resulting in w and a-B ions of this site not being
observed.
w4 x4 y4 z4 w1 x1 y1 z1
B B B B B B Base
A A
O O O O
5' OH
O P O O P O O P O O P O
OH 3' Sugar
−
O O− O− O−
a 1 b 1 c1 d 1 a 4 b 4 c4 d 4
Scheme 3.1 Nomenclature for fragmentation of oligonucleotides.
Peaks which were assigned to a-B and w fragment ions are observed as they were
in low energy CID [68] experiments. Two kinds of fragment ions not seen in low energy
CID are observed in the present study, namely a ions and sugar ring cleavage ions
dubbed Σ. In Scheme 3.2 the proposed cleavage site to form Σ is indicated where the
charge resides on the 5’ fragment. When the charge is on the 3’ fragment, we denote the
fragment ion by ∆. We cannot distinguish between Σ and ∆ for cleavage of a sugar ring in
[d(A)5-2H]2- because these ions have similar masses. Therefore, we did an experiment on
an asymmetric oligonucleotide [d(GCCCC)-2H]2- in which case no ∆ ions were observed.
Hence we assume ∆ ions are not formed from [d(A)5-2H]2- either.
5'
OH
CH2 B
O
H H
H H
∆ O H
−
O P O
O
CH2 B
O
H H
Σ H H
OH H
3'
Scheme 3.2 The proposed sugar ring cleavage sites.
24
_____________________________________________________________
A mechanism for production of a ions based on single-electron loss is proposed in

Scheme 3.3. An ion radical is formed as a result of electron loss from the phosphate group
in a collision with a He atom. A hydrogen atom from the sugar ring is then transferred to
the oxygen radical and the 2’carbon becomes the radical site. This then induces bond
cleavage of the 3’C-O and a double bond is formed between the 2’C and the 3’C
(elimination reaction) and therefore, depending on the position of the charged phosphate
group, a ions and w ions are produced. A mechanism which could lead to production of
sugar ring cleavage ions is more complicated, but a possible mechanism is also based on
initial single-electron loss from the oxygen atom. After the electron loss, again a hydrogen
atom from the sugar ring is transferred to the oxygen radical and a 2’carbon becomes the
radical site. Instead of cleavage of the 3’ C-O bond making a ions and w ions, a 1’C-O
bond cleavage takes place. The O then becomes the radical site and a double bond is
formed between the 1’C and the 2’C. The radical O then induces bond cleavage of the
3’C-4’C bond and the sugar ring is opened as a result of the cleavage of a C-C and a C-O
bond.
O O O O
O P O− O P O− O P O− O P O−
O O O O
CH2 O B CH2 O B CH2 O B CH2 O B O
He
H H H H H H + O P OH
H H H H H O
H H ?H
H H
O H O H O
O P O− O P O O P OH a fragment w fragment
O O O
Scheme 3.3 The proposed mechanism for production of a ions.
The a ions and ring cleavage ions were not observed in low energy CID, but in ion-
ion electron transfer experiments by McLuckey and coworkers [74,75] oligonucleotide
radical ions were formed that decayed along the lines described above. However in both
the present experiments and the experiments of McLuckey, ∆ ions were not observed,
which could be due to the instability of these ions to dissociation before detection.
It appears from Fig. 3.6 that w ions are the dominant fragment ions. This finding is also
different from what is observed in low-energy CID where peaks assigned to w ions have
the same intensity as other fragment ion peaks assigned to a-B or base loss ions. Our
spectra are also different from those of Weimann et al. [73] who performed CID studies at
lab energies around 400 eV and found that the spectra were dominated by low m/q ions
which makes sequencing difficult. But similar behavior was found in ion-ion reaction
experiments [74]. The “excess” w ions would be produced by a single-electron loss
process. The radical reactions lead to a ions, which in principle should produce the same
number of the complementary w ions, as indicated in Scheme 3.3. Dissociation of ∆ ions
can also make w ions. We believe that our high-energy CID spectra, which are dominated
25
_____________________________________________________________
by w ions, make sequencing of oligonucleotides faster and easier than by any other
excitation methods.
The CID spectrum of [d(A)5-4H]4- is shown in Fig. 3.7a. In this spectrum b ions, which
were not seen in low energy CID, are observed. These fragment ions appeared in the CID
spectrum of [d(A)5-3H]3- but were nearly absent in that of [d(A)5-2H]2-. In Fig. 6b a plot of
the relative cross section for b ion formation is shown as a function of the charge state of
the precursor ion. It is evident that b ions are not formed for ions with charge state 2 and
that the cross section increases with charge going from 3 to 4. This charge state
dependence indicates that b ions are formed from ions that have lost two electrons but the
reaction scheme is not understood at present.
a) [−A]
2−
2−
w4
3−
[−A]
w2 2-
a2−B Σ4
a4−B
a3 b4
w1
a4
b3 w3 w4 −
[−A]
0 200 400 600 800 1000 1200 1400 1600

m/q
Cross sections (arb. unit)
1.0 b)
b3
0.8 b4
0.6
0.4
0.2
0.0
1 2 3 4 5
n
Fig.3.7 (a) CID spectrum of [d(A)5-4H]4- in collision with He gas. Note
that b ions are observed in this spectrum. (b) Cross sections for b ion
formation from [d(A)5-nH]n- in He gas as a function of n.
Summary
Electron loss is the dominant reaction channel in high energy (100-250 keV) collisions
between oligonucleotide anions and He. As a consequence of this reaction channel,
fragment peaks which are normally not observed in low-energy CID appear in our
fragment spectra. A general observation is that w ions are much more prominent in the
26
_____________________________________________________________
present high-energy experiments than in low energy CID. We also observed a, sugar ring
cleavage ions dubbed Σ, and b ions. We have proposed a mechanism for the production of
a and Σ ions based on loss of an electron from the precursor ion. Such ions were observed
earlier in ion-ion reaction experiments [74,75]. We suggest that b ions are formed as a
result of double-electron loss.
3.3 Electron attachment to nucleotide monoanions in

collisions with Na: formation of dianions
When high-energy rays such as x-, β, or γ-rays irradiate living cells, genotoxic effects
emerge. Impairment of genome is not induced by direct interaction with the high energy
quanta. Instead it is the secondary species produced by ionization irradiation that destroys
the DNA or RNA molecules. For example, singlet oxygen which is highly reactive and
hence harmful to DNA or RNA can be indirectly produced by UV photon absorption [76].
Free electrons are most abundant among secondary species [77]. Recently, Sanche’s
group [78] used low energy (3 to 20 eV) electrons to irradiate plasmid DNA single strands
and double strands. They found that even when the electron energy is lower than the
ionization energy of nucleobases they observed single strand break (SSB) and double
strand break (DSB). Except for the strand break, transient molecular anions were
observed by dissociated protons being detected which can explain SSB and DSB by low
energy electrons attachment. In order to understand the mechanism of DNA damage by
electron attachment, and since DNA is so large, a reductionist approach has been taken in
the study of electron attachment to isolated nucleobases in vacuum to elucidate the
electronic structure of the anion and to show how Watson-Crick base-pairing is affected
[79-98]. In the following, we move one step further and describe electron binding to
nucleotides.
Formation of dianions
Because a nucleotide carries a negative charge located on the phosphate group, direct
attachment of low kinetic energy electrons (? 0 eV) to anions in vacuum is hindered by a
large Coulomb barrier and is only possible through tunneling. To circumvent this hindrance
we have collided nucleotide monoanions which have high translational kinetic energy (50
keV) with gaseous sodium and looked for electron capture. The idea is that an electron
jump might happen at close approach when the over-the-barrier condition is fulfilled.
A MIKE spectrum obtained for the collision between AMP anions and sodium is
shown in Fig. 3.8b. A spectrum for collisions with neon is included for comparison (Fig.
3.8a). The geometrical cross section of neon is similar to that of sodium but neon's much
larger ionization energy prevents electron transfer. Peaks corresponding to fragment ions
are seen in both spectra. Interestingly a peak at half the m/q value of the parent ion
appears when sodium is used as the collision gas but is absent when neon is used. In
dissociation processes kinetic energy is released resulting in broad peaks in the MIKE
spectra. The peak width of the ion at half the m/q is smaller than that of other peaks, which
implies that this ion is not a fragment ion but instead a dianion. The abundance of the
dianion is about 3% of that of the total abundance of anionic product ions. Hence, in the
collision the anion becomes vibrationally excited and/or an electron is transferred from the
sodium (donor) to the anion (acceptor). Electron detachment results in either neutral or
cationic products. The translational energy of the anions is important for the efficiency of
the electron-transfer process to generate the dianion. In the 30 − 50 keV region, the
27
Electron attachment to nucleotide anions
_____________________________________________________________
abundance of the dianion is the same but decreases at higher energies. At 100 keV the
abundance is roughly two thirds of that at 50 keV. When the collision energy becomes too
high, the interaction time may become too short. Assuming that the anion interacts with a
sodium atom over a distance of 5 −15 Å, the interaction time is 3 − 9 fs (time of passage,
50 keV translational energy), which is shorter than the time of any vibrational period. The
electron transfer is therefore assumed to be vertical. The lifetime of the dianion is at least
of the order of microseconds as the flight time from the collision cell to the detector is 6 µs.
In a similar experiment on the dAMP nucleotide anion (deoxyadenosine
5’−monophosphate, m/q 330) a peak was observed at half the m/q, again owing to the
formation of a dianion (Figure 3.8c), when sodium was the collision gas.
a H2PO4
− AMP
−
−
PO3
−
A −
[AMP−AH]
−
b H2PO4
− AMP
−
PO3
− 2−
A AMP
−
[AMP−AH] [AMP−O]
−
−
[dAMP−AH]
−
c −
H2PO4 dAMP
−
PO3
− A
2−
dAMP
−
[dAMP−O]
80 120 160 200 240 280 320 360
m/q
Fig.3.8 Spectra obtained after collisions between AMP anions and neon (a)
and sodium (b) and between dAMP anions and sodium (c).The inset in (c)
shows the region around half the m/q of the dAMP anion for neon (grey curve)
and sodium (red curve) as collision gases.
What is the electronic structure in the AMP and dAMP dianions? In the gaseous
adenine anion the extra electron is dipole-bound by 12 meV, [82] but the dipole moment of
adenine (2.6 Debye) is extremely close to the critical value for dipole-bound states.[99]
Since the distance between the electron and the adenine molecule is 10 Å, the electron
only slightly affects the structure of adenine. Recent DFT calculations on isolated
nucleobases indicate that Watson−Crick base-pairing stabilizes a covalent anion
28
_____________________________________________________________
structure[88−90] and, based on photoelectron spectroscopy experiments, Hendricks et

al.[87] showed that hydration of the uracil base leads to a transformation from a dipole-
bound anion to a covalent anion. In the AMP dianion a dipole-bound electron experiences
the Coulomb repulsion of the negatively charged phosphate group, which raises the
energy by about 1 eV (14.4 eVÅ/R, R? 10 Å being the distance between the two charges).
Hence it is unlikely that a long-lived, purely dipole-bound state, which is repulsive, exists
for the AMP dianion. Furthermore, because a dipole-bound electron is very sensitive to the
molecular geometry which continuously changes because of atomic motion,
autodetachment within vibrational periods may occur [100]. Rotational motion of the dipole
limits the lifetime as well when the electron begins to slip behind for high rotational
quantum numbers.[100, 101] Most likely the electron goes into the LUMO of the anion
located on the adenine base which is the lowest antibonding π orbital (Figure 3.9a).
a b
Fig.3.9 (a) The LUMO orbital of the AMP anion (b) The SOMO orbital of the AMP dianion.
To understand the electronic structure of the covalent AMP dianion better, we

performed theoretical calculations at the B3LYP6−311++G(2d,p)//PM3 level of theory by
Gaussian [102]. We located a minimum of the dianion with a structure similar to that of the
monoanion (Fig. 3.9b) and found that this dianion is 2.3 eV higher in energy than the
monoanion. For comparison, we calculated that adenine has an EA value of -0.4 eV
(covalent anion), in good agreement with previous calculations [81, 85]. The negative
charge of the phosphate group therefore makes the electron more unbound by 1.9 eV. The
SOMO (singly occupied molecular orbital) of the AMP dianion is similar to the LUMO of the
monoanion though it has diffuse character at the NH2 group (Fig. 3.9b).
Even though a dianion is unstable because of electron autodetachment to the
continuum, the electron may be trapped because of the Coulomb barrier, a phenomenon
previously observed for multiply charged anions [103]. A simple model often employed is
that of an electron in a square well potential at short range and a repulsive Coulomb
potential at long range [104, 105]. However, the large negative binding energy of the AMP
dianion needs some chemical reactions to stabilize.
Hydrogen Loss from Nucleobase Nitrogens

A careful calibration was made on our mass spectrometer. Indeed, a peak which is
located 0.5 m/q below the intact dianions reveals that the AMP dianion is dehydrogenated
dianion after electron capture (Fig. 3.10A). This fragment dianion is the only one that
travels to the detector. Experiments were carried out for other nucleotide anions including
29
_____________________________________________________________
UMP, CMP, GMP, dAMP, dTMP, dCMP, and dGMP, and similar results were found as for
AMP. Previous works have studied electron attachment to the isolated nucleobase, and
hydrogen loss from transient nucleobase anions was indeed observed [82, 106, 107].
Another reaction was dissociation into a neutral nucleobase radical and H−- [78, 108].
However, in our experiments from figure 3.8a and 3.8b, loss of a H– from the transient
dianion was not observed. All these experiments demonstrated that in the presence of low-
energy electrons the nucleobase or DNA backbone is modified due to dissociation along a
[R – H]- coordinate (R denotes the atom from which the hydrogen is lost), but the actual
hydrogen in play still remains to be identified.
−1.5 −1 −0.5 0
∆m/q
Fig.3.10 Narrow scan mass spectra in the region of the dianion formed in
50-keV collisions between nucleotide anions and sodium vapor. The m/q of
the intact dianion formed after electron capture corresponds to 0 on the
axis. The parent ion is AMP (a), AMP-d1 (b), AMP-d2 (c) and AMP-d3 (d).
There are several possibilities e.g. phosphoric acid PO–H, sugar O–H, sugar C–H,
nucleobase N–H, or nucleobase C– H (see Fig. 1.4). Since isolated nucleobases also lose
hydrogen upon electron attachment, it is most likely that the hydrogen lost from the
nucleotide originated on the nucleobase. To determine whether the hydrogen is from a
heteroatom or from carbon we electrosprayed AMP dissolved in CD3OD to selectively
exchange heteroatom hydrogen with deuterium. Ions labelled with one, two or three
deuterium atoms, denoted AMP–d1, AMP–d2 and AMP–d3, were each selected with the
magnet and collided with sodium. It is evident from the MIKE spectra in Fig. 3.10 that a
new peak appears 1 m/q unit below that of the intact dianion peak which implies loss of
deuterium from the transient dianion. Not surprisingly, the ratio of D to H loss increases
with the deuterium content of the parent ion, and D loss is already dominant for the AMP2-
–d3 dianion. These results allow us to conclude that the hydrogen is lost from a
heteroatom and not from a carbon atom. Our experiment does not pinpoint the actual
heteroatom hydrogen. However the fact that isolated nucleobase anions lose hydrogen
30
_____________________________________________________________
indicates that the hydrogen is from the amino group of the adenine. Nucleobases differ in
their number of N–H hydrogens, and comparisons between different nucleotides shed light
on this issue as discussed in the following.
We have determined the cross section for the formation of the dianion for both RNA
and DNA nucleotides. The cross section is a measure of the probability of forming a long-
lived dianion on the microsecond time scale. The results are summarized in Table 3.2 (n is
the number of hydrogens on nucleobase nitrogens), and it is seen that there is a strong
dependence on the chemical structure of the nucleobase. Nucleotides that contain uracil
or thymine have the lowest cross sections whereas the nucleotide with guanine has the
largest. The cross sections for nucleotides that contain either cytosine or adenine are
similar. There is no difference between RNA and DNA nucleotides within the uncertainty of
the experiment which implies that the hydrogen lost from RNA nucleotides is not the C2’-
OH hydrogen since the OH group is substituted by H in DNA nucleotides.
Table 3.2 Cross sections (in Å2) for the formation of [nucleotide – H]2-
Nucleobase n RNA DNA
nucleotide nucleotide
Ura 1 0.10 ? 0.01
Thy 1 0.11 ? 0.01
Cyt 2 0.21 ? 0.03 0.24 ? 0.03
Ade 2 0.26 ? 0.03 0.25 ? 0.03
Gua 3 0.40 ? 0.04 0.34 ? 0.04
0.4 RNA nucleotide

DNA nucleotide
0.3
σ (Å )
2
0.2
0.1
0.0
0 1 2 3
n
Fig.3.11 The cross section σ for formation of the nucleotide
dianion minus a hydrogen atom as a function of the number of
hydrogens on nucleobase nitrogens.
31
_____________________________________________________________
Interestingly, the cross section correlates with the number of N–H hydrogens, n, in
the nucleobase. Indeed, a linear dependence is found when the cross section is plotted as
a function of n (Figure 3.11). Extrapolation to n = 0 predicts a cross section of nearly zero
for a nucleotide composed of a hypothetical nucleobase with no N–H hydrogens. These
two observations indicate that the origin of the lost hydrogen is indeed N–H in the
nucleobase. At the B3LYP/6-311++G(2d,p)/PM3 level of theory we calculate the energy for
vertical attachment of an electron to [AMP – H]- (N-dehydrogenated anion) to be slightly
positive by 0.1 eV, while it is still negative when the lost H is from C2–H or C8–H of adenine.
Our findings are in good agreement with a very recent theoretical paper by Profeta
et al. [109]. They found, based on density functional theory calculations, that the electron
affinity of the thymine radical, thymine minus hydrogen, is significantly larger than that of
thymine and, importantly that the radical with the largest electron affinity is the one that
lacks hydrogen on nitrogen.
A nucleobase anion that lacks hydrogen on nitrogen is a strong base, and when it is
in aqueous solution rapid protonation is expected to occur. Thus, the original nucleobase
is recovered and in principle no damage has occurred. The water content in the interior of
a DNA double helix is, however, limited to about 2.5 water molecules per base pair [110],
and it is therefore interesting to consider how the Watson-Crick base pairing is affected by
the formation of nucleobase anions that lack hydrogen. In other words, the environment in
the interior of the helix is somewhat between bulk solution and vacuum.
The Watson-Crick base pairs are shown in Figure 1.8. The loss of a hydrogen atom
from a certain N-H groups is seen to lower the number of hydrogen bonds between two
bases and hence to a weakening of the selectivity upon which the genetic code is based.
Furthermore, it is evident that [thymine – H] − pairs preferentially with guanine (Figure 3.12)
instead of adenine with possible implications for the genetic code and mutations.
H3C O O N H
H
-
N H N N
N N
O H NH
[Thymine−H]− Guanine
Fig.3.12 Loss of genetic code due to base mispairing after

hydrogen loss from the thymine anion.
Preliminary results of electron attachment to microsolvated nucleotide and

oligonucleotides
A. Microsolvated nucleotide monoanions in collisions with Na
Microsolvated ions are interesting cluster species because they bridge the gas phase and
the solution phase. These ion clusters are fragile so that soft ionization techniques are
needed to bring them into the vacuum. Fenn et al. [111] first observed the clusters of
solute species with electrospray ionization. Since then, much research has been done
32
_____________________________________________________________
[112-118]. For example, solvation shell structures were observed [113, 114] and large
hydrated ions were also found to exhibit bulk solution properties [113].
In our studies, where a hydrogen atom loss from N-H was observed and where this
loss could be one mechanism for DNA damage, it should be studied to what extent water
molecules prevent hydrogen loss from the base? In the following, I present the preliminary
results of electron attachment to microsolvated AMP anions.
Microsolvated AMP–?(H2O)n (n=1∼20) anions are generated by electrospray ionization.
The spray solution was made by dissolving AMP powder in pure methanol (containing
0.1% water). The temperature of the heated capillary was set to 60 oC.
−
AMP ·(H2O)4
a AMP 2−
− H2O
2−
AMP ·(H2O)2
2−
AMP ·H2O
− 4 H2O
− 2 H2O
170 180 190 200

− 3 H2O
120 160 200 240 280 320 360 400

m/z
−
AMP ·13H2O
b +7H2O
+4H2O
2-
AMP
+9H2O
170 180 190 200 210 220 230 240 250 260 270 280 290 300
−
AMP
m/q
0 100 200 300 400 500 600
m/q
Fig.3.13 Electron attachment to microsolvated AMP anions.

(a) AMP–?(H2O)4 (b) AMP–?(H2O)13.
The spectrum for AMP–?(H2O)4 and AMP–?(H2O)13 are shown in Fig. 3.13. The major
peaks are due to water loss from collisionally excited ions, although AMP dianions with
water attached are formed as well. From the intensity distributions of the water loss peaks
of monoanions, the collisional process can be concluded to be statistical [119]. However in
the dianion region not all dianions are observed. Only 1 and 2 water attached dianions
appeared in collisions between AMP–?(H2O)4 and Na. There were nearly no waters
33
_____________________________________________________________
attached to dianions formed in collisions when n is less than 3. When n is larger for
example greater than 13, the dianions with three or four water molecules are absent. One
possible reason is that the water loss from dianions involves a non-statistical mechanism.
AMP dianion and total dianion formation cross sections were measured as a function
of number of water molecules and the results are shown in fig. 3.14. The cross section for
AMP dianion formation increases up to four water molecules and then decreases as more
water molecules are attached. This is because the AMP dianion resulting from hydrated
AMP anion has lower internal energy compared with that from bare AMP anion. However,
for large hydration the energy transferred is not enough to make a bare AMP dianion so
that the cross section decreases. The cross sections for total dianion formation are
increasing with the number of water molecules and the cross section for AMP–?(H2O)16 is a
factor of 20 higher than that for bare AMP–. This is correlated with the second electron
affinities (EA) of AMP. With the attachment of water molecules the possibility of electron
capture to the nucleobase is much higher.
0.9 6
a b
AMP dianion formation cross section (Å )
0.8
2
−
5
Dianion formation cross sections (Å )

AMP .(H2O)n+Na
2
0.7
4
0.6
3
0.5
0.4 2
0.3
1
0.2
0
0 2 4 6 8 10 0 2 4 6 8 10 12 14 16 18
Number of water molecules n Number of water molecules n
Fig.3.14 Formation cross sections for (a) AMP dianions and (b) all dianions.
Table 3.3 Electron binding energies calculated after proton/hydrogen transfer.

Proton/hydrogen Proton/hydrogen EBE Adiabatic EBE vertical Reaction
donor acceptor [eV] [eV]
C1’H N3 -1.0 -0.5 IPT

C2’H C2 0.2 IHT
C2’H N3 -4.3 0.4 IHT
C2’OH N3 -1.6 0.2 IPT
C2’OH C2 0.8 1.0 IPT
C3’OH N3 -0.4 0.3 IPT
POH N7 -2.6 -1.0 IHT
C6NH2 N7 -2.2 -1.1 IHT
No detailed mass calibration was made to determine whether the dianions are intact
or with a hydrogen loss. The second Vertical EA (VEA) of AMP is calculated based on
34
_____________________________________________________________
B3LYP/6-311++G (2d, p)//PM3 level. Up to 4 water molecule AMP clusters, VEA is

negative for the intact anions. However, if internal proton or hydrogen transfer (IPT or IHT)
happens the intact dianion can be stabilized (table 3.3) [120]. From the calculation for
AMP–?(H2O) with H loss from N–H, VEA is found to be 0.2 eV which means the water
molecules can stabilize the dianion by 0.1 eV. When AMP is fully hydrated, the Coulomb
repulsion is reduced a lot and therefore the dipole bound state for adenine can be formed
or the extra electron can also be dipole bound by water clusters so that the hydrogen may
not be lost.
B. electron attachment to oligonucleotide anions

In the next step we collided oligonucleotide anions with Na gas to make an electron
transfer. This study toward a better understanding of DNA damage by low energy
electrons. Nearly no studies of electron attachment to oligonucleotides are carried out
because 1) the source used in the studies by the other groups is the thermo-evaporation
source which can not produce intact oligonucleotide anions or neutrals in the gas phase
and 2) free electrons cannot attach to oligonucleotide anions because of the strong
Coulomb repulsion.
Shown in fig. 3.15 is the MIKE spectrum of [d(A)2–H]– in collisions with Na gas.
Fragment peaks are assigned according to the nomenclature of scheme 3.1. The dianion
peak is clearly seen in the middle of the spectrum. In order to know whether there are
peaks due to electron capture processes the mass spectrum of [d(A)2–H]– in collisions with
neon is required.
1000 −
[dA2−H] +Na
800
w1/d1
−
600
A
Counts
−
400 −A
w1/d1−A
200 2−
[dA2−H]
0
0 100 200 300 400 500 600
m/q
Fig.3.15 MIKE spectrum of dinucleotide anion in collisions with Na.
Recently, Simons et al. [92] proposed a mechanism to explain single strand

breakage (SSB) by low energy electron attachment. In their mechanism the electron is
35
_____________________________________________________________
captured to a π* orbital of the nucleobase. When the sugar-phosphate C–O bond length
exceeds 1.9 Å, the electron can migrate to the σ* orbital of the C–O bond inducing SSB.
Sanche et al. [95] proposed another mechanism in which thermal electrons can attach to a
neutral phosphate P=O bond thus rupturing the DNA. Very recently, Simons et al. [121]
made a calculation showing that zero eV electrons have a low possibility to attach to
phosphate P=O bond. Instead, the electrons with 2∼3 eV energy can attach to P=O bonds
leading to damage. In order to understand which is the electron attachment site, cross
sections for formation of trianions of [d(G)5–2H] 2– and [d(T)5–2H] 2– were measured
(Scheme 3.4). From the studies above the cross sections of mononucleotides containing G
and T have the largest difference when the electron is captured to the nucleobase. Hence,
electron capture to the base still happens for oligonucleotides and then the cross section
difference should be even larger. Otherwise, if the cross sections are independent of the
nucleobase, it suggests that the site for electron attachment is the phosphoric acid P=O.
Indeed, the cross sections we measured for [d(G)5–2H] 2– and [d(T)5–2H] 2– are 2.9 Å2 and
3.5 Å2 respectively. The site of electron attachment to oligonucleotides is the phosphate
group.
e− e− ?
B B e− ?
B
? ?
P S P S P S
Scheme 3.4 Electron capture to mononucleotide (left) and oligonucleotide

(right). B denotes base, P phosphate group and S sugar.
A MIKE spectrum for collisions with Neon is needed for comparison to understand
whether the electron capture to P=O can induce “extra” fragmentations.
Summary for electron attachment to nucleotides

In conclusion, an isolated nucleotide with an extra electron was generated in collisions
between nucleotide anions and sodium vapor and identified with a mass spectrometer.
This is the first time the dianions produced by electron attachment has been detected. The
excess electron is necessarily in a covalent orbital. The dianion is surprisingly long-lived
(microseconds) which makes it a potentially hazardous reagent for chemical reactions. We
also show that electron attachment to nucleotide anions results in loss of hydrogen from
the nucleobase N–H. Dehydrogenation is most likely for guanine, and this nucleobase is
therefore most sensitive to electron bombardment. In an aqueous environment the
[nucleobase–H]– anion is rapidly protonated but when located within a DNA double helix,
where the exposure to water molecules is less, it may survive long enough to cause
severe structural changes in the Watson-Crick base pairs. To judge the importance of this
mutation mechanism, rate constants for proton transfer from water and for base mispairing
are needed. For microsolvated AMP anions the total dianion formation cross sections are
monotonically increasing with the number of water molecules. The missing cluster dianions
indicate there is a non-statistical process involved in the electron capture process. An
36
_____________________________________________________________
excess energy of 1.3 eV is estimated to be transferred into the dianions along with the
electron capture. Trianions of oligonucleotide can also be produced in collisions with Na.
The formation cross sections for trianions of oligonucleotides with different base are
almost identical indicating the capture site for electrons is the phosphoric acid P=O bond.
3.4 Electron capture to peptide cations

Electron capture cross sections by cations in collisions with C60 and Na
The measurements of electron capture and loss is a classical discipline in ion atom
collision studies and information about atomic structure and dynamics can be obtained
from such studies. Recently there has been a growing interest in charge permutation
reactions in collisions between cluster ions or large molecular ions with gases or with
clusters. Electron transfer processes are reasonably well understood for atomic systems
[122] but have not been fully investigated for collisions between more complex systems
such as large molecule- or cluster-ions and atoms, molecules and clusters in the gas
phase. Electron capture by multiply charged ions in collisions with C60 became an active
field of investigation since the pioneering experiments of Walch et al. [123]. For further
references see e.g. Cederquist et al. [59]. The over-the-barrier model has been very
successful in estimating capture cross-sections for such collisions and, recently, it has
been generalized to treat cases with polarizable projectiles and targets of finite geometrical
dimensions [60]. An example here is the successful modeling of target charge state
distributions in slow, non-fragmenting, C60q+ –C60 collisions [60]. Electron capture in
collisions between singly charged alkali clusters and Cs was studied as a function of
cluster size and a strong dependence of the cross-section on the energy defect as well as
on the collision energy was found [124]. Theoretical estimates for these cross-sections
were performed within a microscopic framework called nonadiabatic quantum molecular
dynamics [125], and the importance of the cluster temperature on charge transfer noted.
Fig.3.16 Spectrum obtained for 100 keV collisions between [BK+2H]2+ and Na.
37
Electron capture to peptide cations
_____________________________________________________________
A subgroup of electron transfer processes is the so-called charge inversion

reactions. In these reactions the charge of the molecular ion is changed from positive to
negative or vice versa in collisions with neutral gas atoms or molecules. Since the
electron-transfer processes are nearly vertical, the charge-inverted ions are often highly
vibrationally excited according to the Frank-Condon factors and their fragmentation may
provide structural information. These processes, which are important in molecular mass
spectrometry have recently been reviewed by Danell and Glish [126]. Hayakawa [127] has
described how alkali metal targets can be used in charge inversion reactions to form
negative ions from positive precursor ions.
The present study focuses on measurements of electron capture cross-sections in
collisions between multiply charged peptides or proteins with Na or C60. We find that the
magnitude of the cross-sections depends strongly on the size of the collision partners, on
ionization energies, on projectile charge state and on collision energy. We have applied
the generalized over-the-barrier model to qualitatively account for the measured
dependencies of the geometrical extensions of the collision partners, the target ionization
energy and the projectile charge states. We have, further, deduced approximate values for
the critical distances within which ubiquitin projectiles fragment in collisions with Na and
C60.
Fig.3.17 Spectra obtained for 350 keV collisions between [Ubi +7H]7+ and O2, C60 and Na.
A spectrum showing electron capture in collisions between doubly protonated

bradykinin [BK+2H]2+ and Na is shown in Figure 3.16. The dominant product peak in the
38
_____________________________________________________________
mass spectrum corresponds to [BK+2H]+ but peaks corresponding to collision-induced

dissociation CID are also observed. Our mass resolution is not sufficient for differentiating
between the masses of [BK+2H]+ and [BK+H]+. We can therefore not directly exclude that
the peak corresponds to proton loss instead of electron capture. Control experiment in a
Ne target where this peak is absent however strongly suggests that the peak should be
assigned to electron capture. Electron capture should be much easier in the case of
cations than that of anions because of the strong Coulomb attraction. Bradykinin is a
peptide consisting of nine amino acid residues with a mass of 1062 amu. Spectra showing
electron capture and loss in collisions between seven times protonated ubiquitin [Ubi
+7H]7+ and O2, C60 and Na are shown in Figure 3.17. Ubiquitin is a protein consisting of 76
amino acid residues with a mass of 8.6 kamu. In all three spectra a background originating
from CID is observed. On top of this, electron loss in O2, loss and capture in C60 and
electron capture in Na are observed. Electron loss by cations of amino acid peptides or
proteins in collision with O2 has been dealt with in ref [128,129] and there it was argued
that electron transfer to O2 resulting in O2 plays an important role. The electron loss cross-
section for [Ubi+7H]7+ colliding with O2 was determined to be ∼100 Å2 but only ∼6 Å2 for
collisions with C60. Electron capture reactions are the dominating reaction channels for the
targets Na and C60. Independent of theoretical approach the target ionization potential, the
binding energy of the transferred electron and the relative particle velocity are important
parameters. Since the target atoms (or molecules) are assumed to be in their electronic
ground state before the collision, the ionization potentials of 5.138 eV for Na and 7.6 eV
[130] for C60 were assumed. The projectile ion has a quasi continuum of excited states and
we therefore expect the dominating electron capture process to be near-resonant which
implies that the dependence on collision velocity is weak for low velocities. That was
confirmed from the measured electron capture cross-section versus collision energy for
[Ubi +7H]7+ interacting with Na (Fig. 3.18). It should however be noted that since the
electron transfer process in reality is non-resonant the capture cross section will most
likely approach zero at very low relative velocities. The two targets (Na and C60) also differ
Fig.3.18 Electron capture cross-section for collisions between

[Ubi+7H]7+ and Na as a function of acceleration voltage.
39
_____________________________________________________________
with respect to geometrical size. The radius of the C60 cage structure is 3.5 Å while the Na
atom in this comparison is a point-like particle. The charge state dependence of the
capture and loss cross-sections is illustrated in Figure 3.19, where m/q spectra are shown
for ubiquitin with charge states from 5 to 11 interacting with C60. The cross section for
electron loss is almost independent of charge state (∼6 Å2) while the electron-capture
cross section increases with charge state. In Figure 3.20 is shown the electron-capture
cross section dependence on charge state for collisions between ubiquitin with Na or with
C60. The electron-capture cross section is around a factor of eight larger for interactions
with Na compared to C60, and increases in both cases almost linearly with the charge state
of the projectile ion.
Fig.3.19 Spectra obtained for collisions between protonated

ubiquitin and C60 for charge states from 5 to 11.
40
_____________________________________________________________
350
Na
300 C60
Cross section [Å ]
2
250
200
150
100
50
0
5 6 7 8 9 10 11
Charge state q
Fig.3.20 Electron-captuross sections for collisions between protonated ubiquitin and C60
(circles) or Na (squares) as a function of projectile charge state. Full curve and broken line:
cross sections calculated using the extended over-the-barrier model for electron capture.
To obtain some insight into the electron capture mechanism we have applied the
generalized over-the-barrier model for electron transfer between conducting spherical
objects. The protonated, multiply charged ubiquitin ions are approximated by a conducting
sphere with a radius of 16.0 Å. The estimate of the ubiquitin radius is based on its crystal
structure which is determined by X-ray diffraction analysis. The multiply charged ubiquitin
ions are assumed to possess enough internal energy to allow the excess protons to be
mobile and to move quickly between the basic groups. The ion is, to a good approximation,
spherical and we therefore use a simple conducting sphere model to describe the ions.
Compared to the radius of projectile ions the radius of Na is considered to be 0. So, the
potential which the electron feels at distant r is simplified as
q 1 a a 1 a3
V(r) = e 2 ( − − − + − ) (9)
r R − r Rr Rr − a 2 2 r 2 (r 2 − a 2 )
where R is the distance from Na+ to the center of projectile ions, q is the charge state of
projectile ion and a is the radius of projectile ions. The first two terms of Eq. (9) are the
interactions between the active electron and projectile ions and Na+, the third and fourth
are between the electron and the image charge of Na+ and the last term is the interaction
between the electron and its own image charge. The potential is shown in fig. 3.21 and
has a maximum Vmax at r=rmax. The potential barrier decreases at closer distances. When it
is below the ionization potential of the target gas, the electron can move from the target
gas to the projectile ions. However, the ionization potential is shifted from the initial
potential Ι to a Stark shifted ionization potential Ι* due to the nearby projectile ions.
q 1 a3
I * = I + ∆I , where ∆I = e 2 ( − ) (10)
R 2 R 2 (R 2 − a 2 )
41
_____________________________________________________________
-5
I
-6
∆I
-7
V(r) (eV)
Vmax=I+∆I
-8
-9
-10
16 18 20
r (Å)
Fig.3.21 The potential calculated for the critical distance.
The potential curve shown in fig.3.21 is calculated for R=21.6 Å and q=5. From Eq.
(9) Vmax=−8.13 eV and ∆Ι =−3.01 eV from Eq (10), therefore the Stark shifted ionization
potential Ι* is equal to Vmax then the critical distance Rc between the two particles is 21.6 Å.
Details can be seen in ref. [60]. For C60 as the target the radius is 3.8 Å. The cross-section
for pure electron transfer from the target particle to the projectile ion, σc is defined as
σ c = π(Rc2 − R f2 ) (11)
where Rf is the largest distance at which projectile fragmentation occurs. Calculated Rc
values for collisions between protonated ubiquitin, with charge states n between 5 and 11,
and Na or C60 are given in Table 3.4. The maximum distance between projectile and target
particles at which fragmentation takes place, Rf, was used as a fitting parameter and the
curves shown in Figure 3.20 show the best fit for Rf values of 20.56 Å and 21.79 Å. These
values are reasonable since for collisions between “spherical” ubiquitin and C60, Rf
corresponds to a surface-surface distance of about 2 Å. In such a collision several eV can
be transferred to the ubiquitin ion leading to fragmentation [131]. In comparison, for
collisions with Na the fragmentation distance corresponds to a distance between the
ubiquitin surface and the Na nucleus of around 5 Å. This value is somewhat larger than
expected and could indicate deviation from a spherical shape of the ubiquitin ion. However
the total fragmentation cross section based on this Rf value σf = π20.562 = 1350 Å2 is in
good agreement with cross section values based on drift tube ion mobility spectrometry
[132]. However the ion mobility measurements also showed that the total fragmentation
cross section depended on the charge state of the ubiquitin ion and the use of the same Rf
value for all charge states is questionable. On the other hand, the calculated Rc values
also depend on the size of the ubiquitin ion and therefore size and charge variations
cancel to first order. Again these model calculations are crude and only serve as a
framework for a discussion of the important parameters which enter the description of
42
_____________________________________________________________
electron transfer between such complex ions. We have not discussed to what extent a
conducting sphere is a good model for ubiquitin and C60, but assumed that its use is
justified by its simplicity. Charge transfer cross sections for collisions between various
protonated peptides and Na or C60 are shown in Figure 3.22 as a function of projectile
charge. The conducting sphere model is meaningless here since the projectile
conformation is known to be far from spherical and change from peptide to peptide. In
collisions with C60 the capture cross section increases almost linearly with projectile charge
state and seems not to depend on the type of peptide. For Na as a target the capture
cross-section varies more than a factor of two for a given charge state and the structure or
size of the individual peptide ions seems to play an important role in the electron capture
process.
Table 3.4 The critical distance Rc for collisions between protonated ubiquitin, with charge
states q between 5 and 11and Na or C60.
q Rc (Na) Å Rc (C60) Å
5 21.66 21.95
6 21.84 21.99
7 22.04 22.02
8 22.27 22.05
9 22.52 22.09
10 22.81 22.12
11 23.13 22.17
Fig.3.22 Electron-capture cross sections for collisions between various

protonated peptides and C60 or Na as a function of projectile charge
state. The peptide masses are as follows: M(Arg−Arg−Arg−Arg) = 642
amu, (gramicidin) = 1214 amu, and M(bradykinin) = 1060 amu.
43
_____________________________________________________________
Electron capture induced dissociation of peptide dications

The aim of tandem mass spectrometry (MS/MS) [133,134] experiments is to obtain
information about the structure of molecular ions. As an example amino acid sequence
information of peptides is obtained if the peptide ions undergo reactions where all the
bonds between the constituted amino acids are selectively cleaved with comparable
probabilities in the individual reactions. Collision-induced dissociation (CID) [34,35] is the
conventional sequencing technique which leads to vibrational excitation followed by
intramolecular energy redistribution before fragmentation. The fragment spectra resulting
from CID are normally dominated by b and y ions [135] (Scheme 3.5) resulting from
cleavage of the amide bonds. The fragments produced in low energy CID only reflect
cleavage of the lowest energy bonds and thus do not always give information about the full
amino acid sequence. In high-energy CID a broader range of fragments is observed. More
fragments give more information but also lead to a larger complexity of the spectra and
hence make the interpretation more difficult.
ECD invented by Mclafferty, Zubarev and coworkers also mentioned in Chapter 1,
is an ideal technique which only gives c and z fragments simplifying the interpretation of
mass spectra. In short this technique utilizes a Fourier transform mass spectrometer
where the precursor ions are stored in a Penning ion trap and irradiated by thermal
electrons. After electron–ion recombination the ions fragment and the dominant fragments
are c and z ions stemming from the cleavage of the backbone N–Cα bond. ECD is
believed to be non-ergodic which means that cleavage occurs before the transferred
energy is distributed over the entire molecule. ECD also occurs with a lesser dependence
on the nature of the neighboring amino acid and only the N-side of proline is 100%
resistive to ECD cleavage.
x3 y3 z3 x1 y1 z1
R1 R2 R3 Rn
H2N C CO N C CO N C CO N C COOH
H H H H H H H
a1 b 1 c1 a3 b 3 c3
Scheme 3.5 Nomenclature for fragments of peptides.
Electron capture induced fragmentation in collisions between molecular ions and

gases has been studied by several groups [127,136,137]. Vékey et al. [127] studied, as an
example, decomposition of the benzene dication C6H62+ and found that they could obtain
information about the internal energy of the daughter ion by varying the ionization energy
of the target. The case where a monocation captures two electrons from a target gas
before it fragments belongs to a group of reactions dubbed charge permutation reactions.
Charge reversal ion spectra of polypeptides have provided extra sequence information in
selected cases [127].
I have discussed the mechanism of electron capture to the peptide cations above,
so I here switch to the mass spectrum of fragments in collisions between cations and Ne,
Na and Mg. It is demonstrated that the fragmentation pathways after collisions with Na or
44
_____________________________________________________________
Mg where electron capture plays a dominant role also includes those found in ECD. It
indicates that also electron capture by peptide ions in collisions with atoms with low
ionization energies leads to non-ergodic fragmentation as does capture of free electrons.
This finding was actually predicted by McLafferty [138] before ECD was invented. When
Ne is used as target gas, the fragment spectrum is similar to those obtained in high energy
CID. Based on a naive picture of Na as a Ne atom and a loosely bound electron we have
subtracted the Ne spectrum from the Na spectrum and obtained a spectrum consisting
almost only of a capture peak and peaks that can be associated with c ions. In this way a
simple fragment spectrum that gives the full amino-acid sequence of the peptide in
question is obtained. We suggest that this method could be considered as an attractive
alternative to ECD in single pass experiments where a free electron target is difficult to
implement.
14
Na [M+2H]
+
12
10
6
2+ y10
4 a7 a7 z9
c4 a82+ c10
c6 c7
Intensity
c8 c9
2 a2 c2 a3 a4 a6 a8
0
Ne
12
10
4 c10
2 a4 a7b7 y a z9 x9y10
a2 a3 8 8
0
0 200 400 600 800 1000 1200 1400
m/q
Fig.3.23 CID spectra of Substance P obtained with Na and Ne target gas.
The fragment spectra recorded for doubly protonated amidated Substance P ions
that have collided with Ne or Na atoms are shown in Fig. 3.23. Substance P is a 11-mer
peptide (Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met) which in its doubly protonated
form has a mass of 1348 amu. It has been used in the present investigations as a test
case since its cleavage pattern has been previously studied by ECD. The signal intensity
in Fig. 3.23 is normalized to the target thickness and the intensity of the initial beam. It is
clearly seen that the electron capture peak, [M + 2H]•+ is the dominating one in the Na
spectrum while it is very weak in the Ne spectrum. The electron-capture cross section of
45
_____________________________________________________________
doubly protonated Substance P has been measured in the Na target, and the cross
section for this electron capture reaction is found to be ∼13Å2. In both spectra, peaks that
are characteristic for high energy CID are observed with comparable intensities. It is,
however, also clearly seen that there is a strong enhancement of peaks corresponding to
breaks of the N–Cα bonds the so-called c ions (c6, c7, c8, c9, c10) in the Na spectrum, peaks
14
+
Substance P [M+2H]
12 RPKQQFFGLM
10
Y(Na)-Y(Ne)
4 c 1 c2 c3 c 4 c5 c6 c7 c8c9 c10
0 200 400 600 800 1000 1200 1400

m/q
Fig.3.24 Difference between two spectra of Substance P. The spectrum was
obtained by subtracting the spectrum with Ne from the one with Na.
that are the dominating ones in ECD spectra. The main difference between the two target
gases is the ionization energy, which is 5.14 eV for Na while it is 21.56 eV for Ne. This,
together with the finding of a doubly protonated ion that has captured an electron in Na but
not in Ne, suggests that the difference between the two cases is due to electron capture in
the Na target. In Fig. 3.24 a difference spectrum is shown. This spectrum is very similar to
the fragmentation spectrum obtained for Substance P in ECD. Mainly c ions are found
which facilitates the sequence determination. However, the peaks corresponding to c1 and
c3 are as in ECD, missing. It is well known that ECD is unable to break the amine
backbone bonds on the N-terminal side of proline [36]. The predominance of N-terminal c
ions can, as in ECD, be understood from the amino acid sequence of Substance P. The N-
terminal amino acid is arginine which is likely to carry the charge since it is the most basic
amino acid. The complementary z fragments are hence neutral and can not be detected in
the present experiment.
The fragment spectra recorded for dications of a tryptic decapeptide from signal
recognition particle (SRP) of Saccharomyces cerevisiae that have collided with Ne or Na
atoms are shown in Fig. 3.25. The (SRP) peptide (Ser-Asp-Arg-Glu-Tyr-Pro-Leu-Leu-Ile-
Arg) has in its doubly protonated form a mass of 1262 amu. As for Substance P the two
spectra show several peaks that are characteristic of high energy CID spectra but again
the electron capture product peak, [M + 2H]•+ is the dominating peak in the Na spectrum
while it is very weak in the Ne spectrum. It should also be noted that several peaks which
are characteristic for ECD spectra [139] are observed in the Na case. In contrast to the
case of Substance P both c ions and their complementary z ions are observed. This can
46
_____________________________________________________________
be explained by the presence of two arginine amino acids in the SRP peptide as pos. 3
and 10 one of which is most likely to carry the charge. In the ECD spectrum of SRP in
[139] the dominating peaks correspond to z6+, c7+, c9+ and [M + 2H]•+ ions peaks that are
also prominent in the Na spectrum in the present experiment. We also clearly observe
peaks that can be related to z9 and x9 ions in the Na spectrum. These peaks are almost
absent in the ECD spectrum in [139]. The difference between ECD and our high energy
electron capture-induced fragmentation measurements is not understood at present.
Na
Ne
Fig.3.25 CID spectra of SRP obtained with Na and Ne.
It is also interesting to compare the present SRP (Na) spectrum with a spectrum
obtained with the method “hot electron capture dissociation” (HECD) [139]. In HECD
electrons with an energy of around 10 eV first excite the molecule, then thermalise before
they are captured as low energy electrons. In HECD spectra some of the most abundant
fragments are due to secondary fragmentation caused by the large electron energy which
is transferred to the molecule. In the HECD SRP spectrum a peak corresponding to a z4
fragment that has lost a side chain group from leucine and become a so called w4 ion is a
prominent peak. In the present Na spectrum this peak is missing which indicates that high
energy electron capture-induced fragmentation in an alkali metal target is closer to ECD
than to HECD. This observation is in good accordance with a simple picture of the capture
process where a low energy quasi-free electron is transferred from a loosely bound state
on the Na atom to the dication.
47
_____________________________________________________________
Fig.3.26 Relative intensity of fragment ions of Substance P with Na and Mg

target. The intensities were normalized to the electron capture intensity.
Fig.3.27 Relative intensity of fragment ions of SRP with Na and Mg target

gases. The intensities were normalized to the electron capture intensity.
In order to investigate the role of the ionization energy of the target atom we have
performed fragmentation studies of Substance P and SRP also in a Mg target. The
ionization energy of Mg is 7.64 eV while it is 5.14 eV for Na. In Fig. 3.26 and 3.27 we
compare the relative intensity of fragment ions normalized to the intensity of [M + 2H]+ ions
48
_____________________________________________________________
for both Substance P and SRP in Na and Mg. It should be noted that the relative peak
heights are similar in the two target gases. The correlation factor between the relative
intensities of c and z fragments is 0.86 for both precursor ions. Thus, the binding energy of
the active target electron does not seem to play a major role as long as electron capture is
the dominating reaction channel in the collision. This observation supports the idea about
a non-ergodic character of the electron capture-induced fragmentation process.
Summary
The study of collisionally induced electron transfer in collisions between protonated
proteins or peptides and Na or C60 shows a dependence which is similar to that observed
for atomic species. The cross section for electron transfer between multiply-charged
ubiquitin and Na is as large as 200 Å2 while the total fragmentation cross section is about
1300 Å2 indicating that only glancing collisions leads to electron transfer without
fragmentation. A precise estimate of the electron-capture cross section is difficult to obtain,
but by describing the electron transfer process in the framework of the over-the-barrier
model we have obtained qualitative agreement with measured cross section values for
collisions between multiply charged ubiquitin with Na and C60.
For the studies of fragmentation, we observe cleavage of most backbone amide
bonds. It is argued that the Na spectrum of Substance P can be considered as a sum
spectrum of a high energy CID part and an ECD part. The high energy CID part can be
obtained separately by using Ne as a target and the difference spectrum is almost identical
with an ECD spectrum. The present fragmentation method is thus complementary to ECD
and can be used in beam experiments where low energy electron targets are difficult to
implement.
49
Biomolecular ions in ELISA
_______________________________________________________
Chapter 4

4.1 Decay of isolated biomolecular ions
Canonical and microcanonical ensembles
For a small system in contact with a large system with fixed energy, the small
system is called a canonical system (fig.4.1). For the small system the probability
P for energy E is
Ω(E 0 − E)
P(E) ∝ ρ(E) (1)
Ω(E 0 )
where ρ is the level density of the small system, Ω is the level density of large
system and E0 is the total energy.
We assume that the energy E is a small fraction of the total energy so that
ln Ω(E 0 − E) may be expanded to first order in E,
Ω(E 0 − E) d
ln ≅ 1− E ln Ω(E 0 ) (2)
Ω(E 0 ) dE 0
1
the second term in eq. (2) is equal to , so eq. (1) becomes
k BT
−E
P(E) ∝ ρ(E)e k BT
(3)
After normalization the canonical distribution is obtained
−E
P(E) = ρ(E)e k BT
/Q , (4)
−E
where Q = ∫ ρ(E)e k BT
dE which is also known as the partition function.
So in the canonical distribution the temperature is fixed, but there is a spread in
energy.
The decay rate is given by eq. (5)
ρ(E − E b )
k(E) = v (5)
ρ(E)
where v is the frequency factor and Eb is the binding energy (assuming no
reverse barrier) of a small fragment. This equation can also be derived from
detailed balance, provided that the system is in statistical equilibrium [140-142].
The decay rate as a function of temperature T is
ρ(E)e − E/k BT ρ(E − E b )
k(T) = ∫ dE v (6)
Q ρ(E)
The Arrhenius law in terms of temperature is
50
Decay of isolated biomolecular ions
_______________________________________________________
−Eb
k(T) = ve k BT
(7)
The equation above is derived assuming a canonical ensemble. But for decay of
isolated ions, eq. (7) is not valid because the total energy is conserved not the
temperature.
S(E0!E) few degrees

of freedom
D(E)
other degrees
of freedom
Fig.4.1 (Left) a canonical system in equilibrium with a heat bath, (Right)

a microcanonical system in equilibrium with its own heat bath.
A microcanonical temperature is introduced to overcome this difficulty

[143-147]. A microcanonical ensemble is the ensemble representing a system in
statistical equilibrium at a well defined energy (fig.4.1) and for a system with
excitation energy E and level density ρ(E), the microcanonical temperature Tm is
defined as
1 d
= ln ρ(E) (8)
k BTm dE
in analogy to the definition of the canonical temperature.
To calculate the relation between E and Tm we consider a canonical system
which is in equilibrium with a heat bath at temperature Tm. The average energy
for this system is
∞
ε (Tm ) = ∫ εP(ε )dε (9)
0
−ε
where P(ε( ∝ ρ(ε)e kBTm .

From differentiation of P (ε ) with respect to ε the most probable value ε p is seen
to be equal to the energy E of the system. So what we shall do next is to make
explicitly the relation between ε p and the average energy ε at temperature Tm.
To calculate the integral in eq.(9), the integrand is approximated by a Gaussian in
eq. (10)
51
_______________________________________________________
1
P0 (ε ) = exp(−(ε − ε p ) 2 / 2σ 2 ). (10)
2πσ 2
The width σ is σ = k BC m Tm , where Cm is the derivative of E with respect to Tm,

i.e., Cm is the microcanonical heat capacity.
Because of the skewness of the canonical energy distribution, a skewness factor
S(ε) is introduced
1 d −1
S (ε ) = exp( (ε − ε p ) 3 ( )) (11)
6 dE σ 2
Substituting P(ε) in eq. (9) with eq. (10) and (11), we have
∞
ε (Tm ) = ∫ εP0 (ε )S (ε )dε ≅ E + k BTm (12)
0
We also can have the relation between the canonical (Cc) and microcanonical
heat capacities in eq. (13)
Cc = Cm + k B (13)
From eq. (5) an expansion is made
ρ(E − E b ) d 1
ln ≅ −E b ln ρ(E − E b ) (14)
ρ(E) dE 2
Fig.4.2 Microcanonical caloric curve of tryptophan.
The expansion is evaluated at the average energy before and after the
unimolecular decay. According to the definition of microcanonical temperature
(eq. (8)) we have the decay rate for microcanonical ensembles by substituting
(14) into (8)
52
_______________________________________________________
k(E) = v exp( −E b /k BTe ) (15)
where Te = Tm − E b /2C m is called the effective temperature.
In fig.4.2 the microcanonical caloric curve for tryptophan is shown (see appendix
for details).
The similar formulas were also derived by Klots [148,149] known as finite-heat-
bath theory.
Depletion and 1/t decay law

For an isolated ensemble, the energy distribution g(E) at time 0 is depleted by a
factor exp(-k(E)t) at a later time t. So the emission rate is given by
I(t) = ∫ dEk(E)g(E) exp( −k(E)t) (16)
If g ( E ) = δ ( E − E 0 ) , an exponential emission rate is observed only in this
condition. However if g(E) varies much more slowly than the decay rate it is not
exponential decay any more. Comparing g(E) with k(E)exp(-k(E)t) (also called the
weight function), the latter changes rapidly and we can find the maximum of
weight function from the derivative with respect to E
d
(k(E) exp( −k(E)t)) = 0 .
dE
We have
k' (E)
( − tk' (E))k(E) exp( −k(E)t) = 0
k(E)
showing that the weight function has a maximum at k(E max ) = 1/t . So g(E) can be
pulled out of the integral as a factor g(Emax). Also, the ratio k’/k=dlnk/dE varies
slowly compared to k, and the integral is therefore approximately given by
k k −1 k(E max )
∫ dEk exp( −kt) = ∫ k' k exp( −kt)d ln k = t ∫ k' d(kt) exp( −kt) ≅ t k' (E max )
−1
This leads to eq. (16) as a 1/t decay law

k(E max )
I(t) = t −1 g(E max ) (17)
k' (E max )
According to our assumption, the variation of the last two factors is much slower
than t-1, and the time dependence of the emission rate is therefore close to a t-1
distribution. Tmax is the temperature conresponding to Emax is time dependent
indicating that with increasing time, clusters with lower initial temperatures
dominate the decay. This is a simple decay law. In contrast to the exponential, it
does not contain a characteristic time. The law is also independent of the type of
particle emitted, whether an electron or a massive fragment. However the 1/t
decay law needs an absolute time zero when the ensemble was created.
The depletion of an ensemble of isolated clusters is illustrated in Fig. 4.3. The
main contribution to I(t) comes from a region around the energy Emax, which
decreases slowly with time, and the number of decays within a small time interval
dt is given by the hatched area of width -dEmax.
There are small deviations from the n= −1 power law because of small
contributions to the power of the last two factors in eq.(17).
53
_______________________________________________________
t=0
1.0
0.8
0.6
g(E) 0.4
0.2
t = 1 ms t=10 µs
0.0
10 12 14 16 18 20
Emax(t)
Internal energy (eV)
Fig.4.3 Illustration of depletion at time t of an energy distribution. The hatched

area indicates the additional depletion during a small time interval dt.
For the last factor

k(E max ) 1
=
k' (E max ) d
ln k(E max )
dE max
and inserting k(E max ) ≅ v exp( −E b /k BTmax ) into equation above
2
k(E max ) k BC maxTmax
= ∝ t −δ (18)
k' (E max ) Eb
The heat capacity C is also increasing with temperature but it approaches an
asymptotic value at high temperatures. At temperatures below 1000 K it scales
with a fairly low power of Tmax as C max ∝ Tmax
η
(for example η=0.4 in the case of
2 +η
tryptophan, see fig.4.2). So, we need to know how Tmax scales with time t. A
mathematical trick is applied.
In order to know the power of t we have to calculate the value of the following
d 2 +η
expression, t ln Tmax . For example, if one would like to know how t-1 scales with
dt
d
time t, one calculates t ln t −1 , which gives the value −1.
dt
d 2 +η d 1 1 1 2+η
t ln Tmax = t (2 + η) ln = −(2 + η)t =− (19)
dt dt ln(vt) ln(vt) t G
where G = ln(vt) is the Gspann parameter. Normally G∼20 when t∼10 ms.
So, the last term adds −0.1 to the 1/t decay.
54
_______________________________________________________
Now we consider the variation of power due to g(Emax). In a collision process, the
high-energy tail of the energy distribution is due to scattering with the target gas.
The distribution in energy transferred for a elastic collision is proportional to E−3/2
calculated by assuming the potential is a screened Coulomb potential
proportional to R−2 [150]. The variation for power-law potentials is between an E−2
dependence for Coulomb potential and E−1dependence for a hard-sphere
potential. Applying the same trick to g(Emax) we have
d d ln g(E max ) d ln E max td ln Tmax
t ln g(E max ) = (20)
dt d ln E max d ln Tmax dt
The first term gives −1.5 from the energy distribution after collisions, the second
C(E max )Tmax
term gives is around 2 (for example for tryptophan this value is 1.6
E max
at around 1000 K) and the last term which is known from eq. (19) gives −1/G.
So the total contribution of g(Emax) is around 3/G. it should be noted that g(Emax)
k(E max )
has the opposite sign to . It can be rationalized in the way since g is a
k' (E max )
decreasing function and the argument then decreases with time.
So, including all the corrections the final result is that the power should be very
close to n=−1.
Quenching by radiative cooling

Photon emission is often a competing process. If the hot molecular ions radiate
on the time scale of the decay, their energy decreases and particle emission or
dissociation is quenched nondestructively. A strong deviation from 1/t will appear.
For small ions, emission of a single photon is usually sufficient to quench particle
emission. Normally, the rate of photon emission varies more slowly with
excitation energy than the decay rate and the photon energy is so low that the
effect of emission can be considered as continuous cooling of the molecule. This
is certainly fulfilled for IR radiation from vibrations.
From the assumption above, with radiative cooling the energy E(t) at time t is
given by E(t) = E − Pr t , where Pr is the radiation power.
Now eq.(15) becomes
t
I(t) = ∫ dEk(E(t))g(E) exp( − ∫ k(E(t' ))dt' ) (21)
0
with the Arrhenius law:

Pr t
k(E(t)) = v exp( −E b /k B (Te − )) = v exp( −E b /k BTe ) exp( −t/τ c )
C
1 E P P G
where = b r2 =G r =
τ c k BCTe CTe τ
τ is the cooling time constant.
Inserting into eq.(21)
55
_______________________________________________________
t
I(t) = ∫ dEk(E) exp( −t/τ c )g(E) exp( − ∫ k(E ) exp( −t' /τ c )dt' ) (22)
0
After integrating the last term in eq.(22) we get exp( − τ c k(E)(1 − exp( −t/τ c )) . So
insertion into eq.(22) gives
I(t) = exp( −t/τ c )∫ dEk(E)g(E) exp( − τ c k(E)(1 − exp( −t/τ c )) (23)
1
Again the weight function is strongly peaked at k(E max ) = or
τ c (1 − exp( −t/τ c ))
k BTmax = E b ( ln(vτ c (1 − exp( −t/τ c ))) −1
So the end result for eq. (21) is given by
k(E max ) 1
I(t) ≅ g(E max ) (24)
k' (E max ) τ c ( exp(t/τ c ) − 1)
According to Eq. (24), cooling is not important at short times, t<<τc, so the decay
follows a power law, and the characteristic emission temperature decreases
slowly due to depletion of the hottest clusters by decay. In the opposite limit, t∼τc
depletion becomes unimportant and Tmax approaches a constant value, kBTmax=
Eb/G. However, due to the cooling, the intensity in Eq. (24) decreases nearly
exponentially with a lifetime τc.
Conclusions
For an isolated ensemble the excitation energy is conserved rather than
temperature. As suggested by Bohr and Heisenberg energy and thermodynamic
temperature may be regarded as complementary quantities as in the concept of
position and momentum. In order to use the Arrhenius law to calculate the decay
rate, a microcanonical temperature is introduced. The relation between energy in
the canonical system and energy in the microcanonical system is given by
eq.(12) or the relation between the heat capacities in eq.(13). Now the decay rate
for excitation energy E has a similar formula as the ‘conventional’ Arrhenius
except that the temperature now is replaced by the effective temperature.
For an isolated system the energy distribution cannot be maintained as a
canonical distribution if the molecules dissociate and so the decay of the
ensemble depends on the initial energy. This energy dependence for the decay is
different from the decay of the canonical system, e.g. BIRD experiments in the
rapid energy exchange limit, where all ions decay by a common factor. The
exponential decay can only be observed when the temperature distribution is
narrow in a microcanonical system. In the case where the temperature
distribution is broad the decay follows a power law, e.g. t−n with n∼1. Radiative
emission is a competing channel with fragmentation decay. The power law decay
can be cut off by an exponential decay with a longer time scale due to the
radiative cooling.
Recently, power law decay and radiative cooling have been found in
fullerenes [146,151]. However in the next section, I will show some cases for
amino acids and nucleotides that decay following a power law and for which
radiative cooling is important.
56
Power law decay
_______________________________________________________
4.2 Power law decay of collisionally excited amino

acids and oligonucleotides and radiative cooling
A storage ring is well suited for lifetime measurements because an absolute time
scale is established by the ion injection, and the decay can easily be recorded
turn by turn over several orders of magnitude in time. It covers from the
microsecond revolution time to a storage time of several seconds.
However it can be difficult to have a well-defined excitation energy of the
injected ions. Photoabsorption has a narrow absorption band and therefore may
not results in the desired temperatures. On the other hand in multiphoton
absorption, electron impact or collisional activation process there is a
considerable spread in excitation energy. Moreover, most conventional ion
sources such as sputter sources or plasma sources produce ions with ill-defined
internal excitation. It is therefore of interest to investigate what information can be
obtained from measurements on ions with a broad distribution in internal energy.
As discussed above with a wide-spread in the internal energy distribution the
decay follows a 1/t law and radiative cooling from vibrational modes competes
with the decay. When the decay is faster than radiative cooling, the characteristic
quenching time τc can be observed. Therefore for longer lifetime decay, it can
only be studied in the condition where the internal energy distribution is
maintained either by photoabsorption or emission [38.39,152,153] or gas
collisions [154].
Next I will show examples first from amino acids followed by results from
oligonucleotides.
Ne gas
Fig.4.4 A gas inlet is mounted after the ion trap. Ions can
collide with gas when extracted to the Einzel lens.
57
_______________________________________________________
Power law decay of amino acid cations

In order to produce a broad spectrum of excitation energies we introduced Ne
gas into the last chamber, as illustrated in the fig.4.4. We also tried heavier gases,
Ar and Xe, but they were found to be less effective. After extraction the ions were
accelerated towards the Einzel lens with a potential on the first lens of about
−300 V, and during this acceleration they may collide with Ne atoms. Even for the
heaviest ions, tryptophan with mass number 205, the centre-of-mass energy in
collisions with Ne can be up to 25 eV, and the collisions should therefore lead to
a long tail in the distribution in internal energy where the decay should be faster
than the radiative cooling and thus the quenching time τc may be measured.
The neutral fragments originate partly from decay of metastable ions, partly
from collisions with the rest gas in the ring. Gas collisions dominate at long times,
as illustrated by the spectrum in Fig. 4.5, recorded with an injection rate of 0.05
Hz. Metastable ions, produced after collisions in the source, decay within several
fractions of a second and the rest of the spectrum shows an exponential decay
due to depletion of the ion bunch by collisions with residual gas with a lifetime of
9.3 s. But in the following experiments we only record the spectrum up to 80 ms
and therefore the depletion due to collisions with the residual gas in ELISA is
negligible and neutrals count rate from gas collisions can be considered as a
constant.
Fig.4.5 Time spectrum for 20 s storage of protonated tyrosine. Except for the
first fraction of a second, the yield of neutrals is dominated by dissociation
induced by collisions with the rest gas, and the exponential decrease of the
signal corresponds to the reduction of the number of stored ions.
58
Power law decay
_______________________________________________________
In order to show the power law decay more explicitly, a log-log plot is used
to show the spectrum and a semilog plot to illustrate the details of the spectrum.
In fig.4.6 the spectrum for phenylalanine is shown both in a log-log plot and in a
semilog plot. The logarithmic time scale is useful for observation of the power-law
behavior, and up to about 10 ms the yield follows closely the 1/t dependence
given by the dashed curve. The yield is then reduced by radiative cooling and, as
illustrated more clearly with the linear time scale, it approaches a constant value
corresponding to fragmentation by gas collisions.
Fig.4.6 Time spectrum for 100 ms storage of protonated phenylalanine. The dashed line in
the log-log plot in the left panel is proportional to 1/t while the solid curve is a fit with the
expression in equation (25) with the parameters given in Table 1. A semilog plot is shown
in the right panel.
Based on eq.(24) the spectrum is fitted with the function,

t 1
I(t) = N( ) δ t/τ c +K (25)
τ c e −1
t
where N is a normalization constant and the factor ( )δ corrects the effective
τc
power in the power law decay due to the first two factors of eq.(24). K is the
background due to collisions with the residual gas. So, the most important
parameter τc can be deduced from the experiments. In fig.4.7 the spectra for the
three typical amino acids discussed in chapter 1 are shown and the fitting
parameters together with those for phenylalanine are listed in table 1.
Table 1 fitting parameters for eq.(25)
Molecule δ τc (ms)
Phe+H+ 0.02 13
Trp+H+ 0.03 17
Lys+H+ -0.05 13
Gly+H+ 0.13 15
59
_______________________________________________________
Time (ms)
Fig.4.7 As Fig. 4.6 but for tryptophan (top), lysine (middle), and glycine (bottom).
60
Power law decay
_______________________________________________________
The amino acids containing an aromatic ring show similar results with the power
close to unity while for lysine with a long linear side chain the slope is slightly
steeper and for smallest amino acid glycine the slope is less steep. As discussed
in section 4.1, the power should be close to 1 but in the case of glycine this
power somewhat deviates from 1. This deviation may result from the competing
fragmentation channels. It is well-known from studies of collision induced
dissociation of biomolecules that usually there are many competing
fragmentation channels, and that the relative abundance of fragment ions
depends on the collision energy [155]. So the Arrhenius parameters can be quite
different for different channels. Suppose the rate constants for two fragmentation
channels are equal at some excitation energy, the channel with the higher
activation energy will dominate for higher excitation energies and the one with
the lower activation energy (and smaller frequency ν) will take over at lower
excitation energies which leads to the neutral rate increasing at longer time thus
the logarithmic slope will be reduced (see ref.[144]). Experiments have been
done on tyrosine (with aromatic side chain), arginine (with linear side chain) and
alanine (with methyl side chain). Similar results were observed. However the
quenching times τc lie between 10 and 20 ms.
For the radiative cooling, we approximate the vibrations by one-dimensional
harmonic oscillators. Classically the power radiated from a charge q oscillating
with frequency ω is given by
2ω 4 q 2 < x 2 >
Pr =
3c 3
in Gaussian units. We can adapt it to a quantum system by replacing <x2> with
E/mω2. This leads to the power radiated at temperature T,
2ω 2 q 2 hω
Pr = (26)
3Mc exp( hω/k BT) − 1
3
where the last factor is the average excitation energy. Since the radiated power
is proportional to this quantity, the energy will decrease exponentially for an
isolated oscillator. The formula also shows the freeze-out of high-frequency
transitions at low temperatures.
As an example we have carried out calculations for protonated tryptophan.
The structure was optimized at the B3LYP/6-31+G(d) level of density functional
theory with the Gaussian 98 package. All calculated frequencies are real, which
confirms that the stationary point is a local minimum on the potential energy
surface. The resulting caloric curve was already presented in Fig4.2. In addition
to the frequencies also the IR intensities were calculated from the dipole
derivatives, which replace the square root of the factor q2/M in equation (26).
However since the units of q2/M are in km/mole, a unit conversion has been
applied to change to Gaussian units. The whole calculation process is given in
the appendix D for further reference. Based on the microcanonical caloric curve,
we calculate the cooling rate defined as Pr/CTe in eq. (22) and the relation
between cooling lifetime τ and quenching time τc is τ=Gτc. In fig.4.8 the
calculated radiative cooling rate is shown. The calculated value can be compared
with the value determined by experiments when the Gspann parameter and
61
_______________________________________________________
activation energy are known. We observed that the dominant decay channel for
tryptophan is the loss of a side chain. So we performed the calculation again for
the primary ions and also for the fragments. The calculation level is at B3LYP/6-
311+G(2d,p) level and corrected for zero-point motion [non-scaled B3LYP/6-
31+G(d) values]. The activation energy for this fragmentation channel is Ed=1.5
eV. In order to get the frequency factor an estimation was made on the decay
rate for a well defined energy from a measurement of laser induced
fragmentation in ELISA. We measure that the lifetime is in the range 5 ∼15 µs.
From observation of a linear dependence on laser power we conclude that the
signal results from absorption of a single photon from fourth harmonic photons,
which gives a total excitation energy of 4.95 eV, including the thermal energy at
room temperature. The frequency factor in the Arrhenius formula is then
calculated to be v ∼ 2 × 1011 s−1 [156]. So we obtained for the Gspann parameter
at 17 ms, G=ln(νt) ∼22. The effective temperature is obtained by Te = Ed/kBG ∼
800 K corresponding to an initial microcanonical temperature of 950 K.
Temperature (K)
Fig.4.8 Calculated radiative cooling rate Pr/CT for protonated tryptophan, compared
with the rate derived from the measured quenching lifetime given in Table 1.
With these parameters the cooling rate has been calculated from our measured
quenching rate and, as shown in Fig.4.8, there is excellent agreement with the
theoretical calculation. The experimental uncertainty is indicated by the error bar
but there is also a considerable uncertainty in the theoretical calculation,
stemming mainly from the evaluation of the dipole derivatives. It is difficult to
calculate the accurate value of IR intensities even though a high level calculation
was used for small molecules. For example Nielsen et. al. [157] applied
B3LYP/LANL2DZ1+diffuse level to calculate the infrared spectrum of Br−HCHBr.
62
Power law decay
_______________________________________________________
The agreement with experiments in some cases is 4 or 5 times less than the
experiments. When the molecules are larger, the ab initio calculation is too time-
consuming and semiempirical methods have to be applied instead. Price and
Williams [158] multiplied dipole derivative by a factor of 3 from AM1 calculations
which was based on the comparison with calculations from RHF and MP2
method tending to give 2.5 times lower than the experiments [159,160]. However
they concluded it was not known if AM1 transition dipole multiplication factor was
valid for larger ions. Moreover, for big molecules in the potential energy surface a
large number of local minima exist which correspond to different conformations.
The calculation thus may end at some local minima which has a quite different
dipole derivative. Concerning the discussion above the method applied to
calculate the radiative power is rather crude and can only give information on the
magnitude of cooling lifetime.
Power law decay of oligonucleotide anions

Experiments have also been done on oligonucleotide anions. The major
fragmentation channel is loss of a nucleobase as discussed in chapter 2.
Williams and coworkers [38] measured the activation energy and preexponential
factor for this fragmentation channel which was 1.03 eV and 1010.3 s−1
respectively (loss of neutral adenine). So the major difference from the amino
acids is that the activation energy is lower (1.5 eV for tryptophan dissociation).
For an ensemble with a broad energy distribution the radiative cooling starts to
compete with decay at the effective temperature, Te=Ed/kBG. When the molecule
is big, the effective temperature approaches its corresponding microcanonical
temperature. This temperature depends on two factors, the activation energy Ed
and Gspann parameter. For a tight transition state the frequency factor is around
1011 s−1, and G is around 20. Changing the quenching time τc from 10 ms to 30
ms can only vary G about 5%. So the major determining factor of temperature is
the activation energy Ed.
In the case of oligonucleotides at the temperature, where cooling can
compete with decay, is then around 550 K which is far lower than that for amino
acids. Above this temperature the decay follows a power law but when the decay
reaches this temperature, the cooling starts to quench the decay. So the
quenching time τc for oligonucleotides is somewhat longer than that for amino
acids (15 ms). On the other hand, if the activation energy is higher (e.g. 2 eV) for
a certain system, the initial temperature for cooling should be much higher than
for amino acids and the quenching time should be shorter than 15 ms of amino
acids. This is true in the case of electron emission after laser excitation of C60−
[161]. The electron binding energy of the C60 anion is 2.67 eV, so the initial
temperature for cooling is about 1500 K and the characteristic quenching time is
determined to be 5.5 ms which is shorter than the 15 ms for amino acids.
The spectrum of [AMP−H]− is shown in fig.4.9. Two fitting functions are
applied. One is eq.(25) including quenching time τc (red curve) and other is the
normal power law fitting (blue curve). For the red curve in fig.4.9 τc is 25 ms and
the power is −0.98 which is the same power as for the blue curve. These curves
are almost identical except that there is small difference at longer time range in
63
_______________________________________________________
the spectrum. However after 25 ms, background signals from residual gas
dominate so that the quenching time is ill-defined. It is consistent with the
discussion above that the quenching time of oligonucleotides should be longer
than that of amino acids.
10000 −
[AMP−H]
1000
Counts
100
10
0.1 1 10 100
Time (ms)
Fig.4.9 Time spectrum for AMP anion for the 100 ms storage time.
Red curve is a fit with eq. (25) with a quenching time and blue curve
fits with simple power law decay.
Experiments have also been carried out on the same oligonucleotides with
different charge states and the spectra are shown in fig.4.10. The idea behind is
to obtain activation energy of weak fragmentation channel. It will be explained in
the following:
For a weak decay channel kW <<ks where kW is the decay rate for the weak
channel and ks is the decay rate for the strong channel. However, if only the
signal from the weak decay channel can be observed, the intensity is now given
by
I(t) = ∫ dEk w (E) exp( −k s t)g(E) (27)
Apply the Arrhenius law again
k w = v w exp( −E w /k BT), k s = v s exp( −E s /k BT)
and we have k w = v w (k s /v s ) Ew /Es where Ew and Es are the activation energies of
weak and strong decays respectively.
Insert into eq.(27) with µ = E w /E s
I(t) ∝ ∫ dEk s exp( −k s t)g(E) which is equal to
µ
64
Power law decay
_______________________________________________________
k s (E m ) µ µ −1 '
k' s (E m ) ∫
t − µ g(E m ) t k s k s exp( −k s t)dE (28)
1 d 1
(tk s ) µ , so the integral in eq.(28) becomes ∫ dx exp( − x 1/µ )
µ −1
while t µ k s k s' =
µ dE µ
independent of time t.
k (E )
Finally, the intensity I(t) ∝ t − µ g(E m ) s m (29)
k' s (E m )
d
Em now is determined by (k sµ exp( −k s t)) = 0 which gives k s (E m ) = µ/t .
dE
From eq.(29) we have E s = E w /µ .
1000 2−
[d(A)5−2H]
100
Power = −0.98
10
Counts
3−
[d(A)5−3H]
1000
100
Power = −0.97
10
0.1 1 10 100
Time (ms)
Fig.4.10 Time spectrum of 100 ms storage for oligonucleotide d(A)5

anions with dianion (top) and trianion (bottom). Red curve is the fit
with simple power law decay.
In the case of oligonucleotide fragmentation, the loss of a neutral base
competes with loss of a charged base depending on the charge state of the
primary ions [69,70]. For example, in the dissociation of [d(A)5−2H]2− loss of
neutral adenine dominates while for [d(A)5−3H]3− loss of charged adenine
dominated. From studying the power of [d(A)5−3H]3− decay we can obtain the
65
_______________________________________________________
activation energy for loss of charged base (1.03/µ). The measured power −0.97
for [d(A)5−3H]3− indicates that the activation energy difference between loss of
neutral and charged base is quite small. It has been applied to obtain the
activation energy of C2 emission from excited C60 [162].
Summary
We have performed measurements on molecules with a broad distribution in
excitation energy, and have chosen representative examples of amino acid
cations and nucleotide anions. The results confirm the approximate 1/t decay law
which was observed for clusters. A strong reduction of power law decay is also
observed for biomolecules which can be explained by radiative cooling. The
quenching time strongly depends on the activation energy for dissociation. We
measured around 15 ms for amino acids and >20 ms for nucleotides. The
observed power-law spectra for a time shorter than τc give indications of a
temperature dependence on competition between different fragmentation
channels. The weak channel decay measurement for [d(A)5−3H]3− shows the
activation energy for charged base loss is similar to the neutral base loss
because a power of unity is nearly observed.
However the power law decay diminishes all the characteristics of
dissociation, e.g., the Arrhenius parameters can not be determined. In the next
section, I will discuss the decays of some examples with the well-defined internal
energy. Arrhenius parameters can be deduced. Other processes like dissociation
via nonergoidc process can also be studied.
4.3 Photodissociation of protonated nucleotides

and peptides
If the initial energy is well defined, the excitation energy after photon absorption
is also known. This is an advantage compared with methods based on excitation
in collisions with other molecules or with a surface, e.g. threshold measurements
of collision-induced dissociation [163].
The BIRD technique has been used successfully to study dissociation
kinetics. Depending on the size of the molecules studied, it can roughly be
divided into three regimes. For large biomolecules including both proteins and
oligonucleotides, the decay temperature of the molecules is equal to the
temperature of the surrounding walls in the rapid energy exchange (REX) limit
where the rate of absorption and emission of infrared photons is higher than the
dissociation rate. Thus the obtained Arrhenius parameters, e.g., Ebobs, are equal
to real dissociation kinetic parameters, e.g. Eb? . For small molecules the decay is
far faster than the absorption of infrared photons, so Dunbar proposed a method
to relate Ebobs with Eb? [164]. But for middle-sized molecules the system is quite
complex, e.g. a set of master equations are established to resolve the
unimolecular decay rate as a function of temperature [165]. Pointed out, it is
easier to study large molecules because the observed parameters are equal to
the real Arrhenius parameters. However the problem how large is large is still
66
Photodissociation of protonated nucleotides and peptides
_______________________________________________________
unresolved. For example, it was shown that for ion masses greater than 1.6 kDa,
the measured BIRD kinetics will be in the REX limit at temperatures below 520 K
[158]. But Dunbar had a mass limit three times smaller [153]. The key to
understanding is the relative rate between the decay and photon exchange. The
BIRD technique and other thermal-dissociation techniques are also limited to
fairly low temperatures (normally 400 to 500 K). So if the ions are stable within
this temperature range, there is no dissociation at all [38]. For dissociation with
many competing pathways, the processes with low activation energy and fairly
small Arrhenius pre-factor may at high temperatures be replaced by more direct
processes with higher activation energies and larger pre-factors. Measurements
at the higher temperatures obtained by laser excitation are therefore useful to
obtain a more complete picture of the statistical dissociation kinetics. They
complement the BIRD experiments. In addition, there may be direct (non-
ergodic) fragmentation channels after electronic excitation.
Photodissociation of protonated oligonucleotides with different lengths

The nucleobases strongly absorb ultraviolet (UV) radiation with a maximum at
260 nm. The S0 ? S1 transition is due to excitations to π* orbitals [166,167]. The
photophysics of nucleobases and nucleotides has been studied extensively [168-
171]. After photoexcitation of the adenosine nucleoside (no phosphate group,
abbreviated Ado) at 263 nm, internal conversion from the excited state (S1) to the
electronic ground state (S0) has been shown to occur with a time constant of 290
fs [168]. The fluorescence quantum yield is very small, 10−4 or less [172], and
intersystem crossing to a lower-lying triplet state is negligible [169]. Initially the
vibrational excitation energy is deposited in the adenine base but intramolecular
vibrational redistribution (IVR) is assumed to occur rapidly [168]. Vibrational
cooling of Ado by solvent interactions then occurs in less than 2 ps [168]. It is
unknown whether photoproduct formation competes with the energy dissipation
and whether prompt processes are of significance. Prompt processes would be
especially hazardous for a biological system if they were faster than solvent
quenching of the excess energy. To address these issues the lifetime of the
unsolvated molecule must be measured.
Photoexcitation was performed at 266 nm. The source of 266-nm photons
was the fourth harmonic of a Nd:YAG laser (Quanta-Ray, Spectra-Physics) also
operated at 10 Hz. The fundamental of 1064 nm was frequency doubled to 532
nm and again doubled to 266 nm. Again for lifetime measurements, a storage
time of 40 ms or more before irradiation is necessary to allow time for the decay
of ions which have been excited in gas collisions during acceleration and
injection. The irradiation resulted in a large production of neutral particles after
bond dissociation (shown for the cation in Fig. 4.11). Again only neutrals formed
in the injection side were counted by the detector.
The decay spectra after laser excitation of four different oligonucleotide
monocations ([d(A)2+H]+, [d(A)3+H]+, [d(A)4+H]+ and [d(A)5+H]+) are shown in Fig.
4.12. The red curves are for fitting with exponential decays. [d(A)2+H]+ has only
one component while the other three have two components. However for
67
_______________________________________________________
[d(A)2+H]+ one exponential does not represent the data well. The blue curve
fitting will be explained in the following.
+
[d(A)2+H]
Counts
0 20 40 60 80
Time (ms)
Fig.4.11 Irradiation of dinucleotide cations [d(A)2+H]+ in ELISA. The initial count rate
after injection is due to the decay of ‘‘hot’’ ions, but after ca. 20 ms the signal is
dominated by collisional decay. Laser excitation after 60 ms of storage time causes
a large increase in the count rate. The depletion of the ion beam is reflected in a
lower rate of collisional decay after laser excitation than before.
As discussed in section 4.1, the decay can only have an exponential
function when the initial energy distribution is narrow, e.g., a Dirac delta function.
In the experiments the ions are in equilibrium with the ion trap before being
extracted so the energy distribution is approximated by a Gaussian distribution
given by
⎛ (T − Tex )2 ⎞ C0 k B
g (T ) ∝ exp⎜⎜ − ⎟ where δ =
⎟ T0 with ion trap temperature T0.
⎝ 2δ 2
⎠ C
The decay rate is given by
I (t ) = ∫ k (T )g (T ) exp(− k (T )t )dT (30)
Because of distribution of the energy, the neutral counting rate will increase at
longer time so that a small deviation from exponential decay will be observed. It
can be understood the same way as two fragmentation channels competing with
each other with different Arrhenius parameters as discussed in section 4.2. The
blue curve is from fitting to eq.(30) which is quite well representative of the data.
For the spectra of the other three, one-exponential fitting with eq.(30) can not
represent the data because the deviation from one exponential is quite large, and
therefore a second component is introduced. In table 2 the fitting parameters are
listed for the four oligonucleotide cations.
68
_______________________________________________________
+
100000 + [d(A)3+H]
[d(A)2+H]
1000
10000
Counts
1000
100
100
0 1 2 3 4 5 0 5 10 15 20 25 30 35 40
Time (ms) Time (ms)
100000 + +
[d(A)4+H] [d(A)5+H]
10000
10000
1000
Counts
1000
100
100 10
0 8 16 5 10 15 20 25 30
Time (ms) Time (ms)
Fig. 4.12 Decay spectra of different oligonucleotide cations after laser excitation. See text for details
69
_______________________________________________________
Table 2. Measured lifetimes (in ms) for different nucleotides [d(A)n+H]+.
n 2 3 4 5
First component 0.025 0.554 0.043 0.245
# of photons 1 1 2 2
Scond component 3.65 3.32 1.02
# of photons 1 1 2
+
[d(A)3+H]
Ratio between two components (arb. unit)
+
[d(A)4+H]
+
[d(A)5+H]
0
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9
Pulse energy (mJ)
Fig.4.13 the ratio between the two components after

photoexcitation as a function of pulse energy.
In order to find the temperature after photoabsorption it would be better to

measure the neutral yields as a function of laser power to determine how many
photons are absorbed. However in order to change the laser power we have to
change the power of the flash lamp of the Nd:YAG laser, which leads to variation
of the size of the laser spots especially when the power of the flash lamp is quite
small. Therefore plotting the yield as a function of laser power sometimes fails to
give information about how many photons are absorbed. For a decay spectrum
70
_______________________________________________________
with multicomponents, instead of plotting the yields as a function of laser power
individually for each component, we plot the ratio between the different
components as a function of laser power which then gives information on the
relative number of photon absorbed. In Fig.4.13 the ratios of the two components
for the three oligonucleotides are shown. In the case of d(A)3 and d(A)5, the ratio
is independent of laser power indicating that the two components are due to the
same number of photons, while for d(A)4 the ratio is linearly increasing which
indicates that the slow component has absorbed one photon less than the fast
component. The slow component for d(A)3 and d(A)5 could be due to an
intersystem crossing between S1 and the lower-lying triplet state (T1). The triplet-
state ions are long lived as the intersystem crossing to the electronic ground
state is spin forbidden, which slows the decay by an order of magnitude.
40
Microcanonical caloric curves
35 d(A)5
30
d(A)4
Energy (eV)
25
20
d(A)3
15
d(A)2
10
0
0 200 400 600 800 1000 1200 1400
Microcanonical temperature (K)
Fig.4.14 Microcanonical caloric curve of nucleotides with different sizes.
Concerning the lifetimes in table 2 and the relative numbers of photons

absorbed, we can estimate the absolute number of photons absorbed. A simple
approach is taken in the following. Roughly the heat capacity of oligonucleotides
with different lengths is proportional to their length, so the heat capacity of d(A)2
is about half that of d(A)4. d(A)2 and the fast component of d(A)4 have similar
lifetime, and they therefore have a similar final temperature after photoabsorption.
Hence the numbers of photons absorbed by d(A)4 should be twice that of d(A)2.
For example, if d(A)2 absorbs one photon then d(A)4 absorbs two (the slow
component of d(A)4 is then due to one photon absorbed). In this way the fast
components of d(A)3 and d(A)5 can be argued to absorb one and two photons,
respectively. On the other hand if we instead assume that d(A)2 absorbs two
photons and d(A)4 (fast component) absorbs four, then the slow component of
71
_______________________________________________________
d(A)4 absorbs three photons. The lifetime of d(A)3 (fast component) is shorter
than that of d(A)4 (slow component), so the temperature of d(A)3 is higher than
that of d(A)4 (slow component). The only way for d(A)3 at higher temperature is to
absorb more than three photons. If so, the lifetime of d(A)3 should be similar to or
shorter than that for d(A)2. It is contradictory to the experimental results. Hence
the first approach is most likely and the number of photons absorbed is
summarized in Table 2.
11 d(A)2
d(A)4
10
9
d(A)5
8
lnk
d(A)3
7
d(A)4
second component
6
5
14 15 16 17 18 19 20
1/kBT
Fig.4.15 Arrhenius plot for nucleotide cations. Solid line for fitting with four fast
components and dashed line for fitting including the slow component of d(A)4.
Next we perform a semiempirical PM3 level calculation to obtain the

vibrational frequencies. The microcanonical caloric curves and heat capacities
can be calculated (see details in appendix). In fig. 4.14 the microcanonical caloric
curve is shown. Knowing the number of photons absorbed, the final temperature
can be calculated. Then we can make an Arrhenius plot. A small correction has
to be made to the Arrhenius decay formula because of the finite-heat-bath.
Eb E 1
k = v exp( ) . So ln k = ln v − b ( ) . Because the last term is
Eb k BT Eb
k B (T − ) 1−
2C 2CT
only a small correction for large molecules, an expansion is made for the last
term
E E
ln k = ln v − b (1 + b ) (31)
k BT 2CT
72
_______________________________________________________
A fit is made according to eq.(31) shown in fig.4.15. The solid line is for only
fitting with the fast components of the four nucleotides because for the slow
component of d(A)4 there can be some decay from a triplet state component after
2-photon absorption. Therefore a fit to the points give Eb=0.88 eV and ν=1010.2 s-1.
If we include the slow component in the fitting (dashed line), the activation energy
and frequency factor are then 0.80 eV and 109.6 s-1, respectively. The two fits are
quite similar.
The Arrhenius parameters obtained from BIRD experiments [38] were
determined to be Eb=1.03 eV and ν=1010.3 s-1 for the loss of neutral base. The
agreement with our high-temperature date is quite good. It should be noted that
BIRD experiments studied the dissociation of oligonucleotide anions while we
study cations and a lower dissociation energy is expected for the cations. This
was explained by Wang et al. [71]. They studied the difference between
oligonucleotide cation and anion fragmentation pattern under the same
experimental conditions concluding that protonation of bases may increase the
chance for cleavage of the glycosidic linkage and this could be due to a lower
activation energy.
Using the Arrhenius parameters obtained in this experiment, we obtain that
the lifetime of the AMP cation is around 2∼3 µs which is shorter than that for AMP
cations measured before (16? 6 µs) [173]. There are some explanations for this.
The lifetime calculated is highly sensitive to the Arrhenius parameters. For
example, if the frequency factor 109.6 is used instead of 1010.2, the calculated
lifetime is then 10 µs which is similar to that for the measurement. Another
explanation is that the 16 µs lifetime observed in the experiment is in fact due to
the decay from triplet state to singlet state and not the statistical dissociation
lifetime (2∼3 µs).
Until now we have considered only statistical processes. However, an
interesting question arises as to whether the ion beam depletion (fig.4.11) is
completely accounted for by ions decaying via an ergodic process. The time
constant for a nonergodic process is much smaller than the revolution time in the
storage ring, likely less than a picosecond (vide infra). Thus, a nonergodic
process will cause a depletion of the ion beam but will not contribute to the
neutrals count rate in our experiment.
In the following we estimate the ion beam depletion associated with the
observed decay. The rate of neutrals formed in collisions is an exponential
dN N 0
n=− = exp( −t / τ )
dt τ
where N and N0 are the number of ions at time t and time zero, respectively, and
τ is the collisional lifetime. Similarly, the rate of neutrals formed after laser
excitation of N0* ions with lifetime τ* is given by
*
dN * N 0
n* = − = * exp( −t/τ * )
dt τ
From the ratio of these two rates the proportion of depleted ions is
73
_______________________________________________________
Total
Pure statistical process (×20)
Depletion
Total
Pure statistical process (×7)
Depletion
0.0 0.2 0.4 0.6 0.8 1.0

Pulse energy (mJ)
Fig.4.16 Depletion of the ion beam as a function of laser

pulse energy for d(A)2 (top) and d(A)5 (bottom).
74
_______________________________________________________
N 0* n * τ * n* τ *
depletion = = exp(t/τ * − t/τ ) ≈ exp(t/τ * ) (32)
N0 n τ n τ
The approximation is made since the collisional lifetime is far longer than the time
for decay after laser activation (τ>>τ*). The depletion determined by eq.(32) only
accounts for the dissociation from the ergodic process. The total depletion is
given by
n − nL
depletion t = ,
n
where nL is the neutrals counts rate at several tens of milliseconds after laser
excitation.
In fig.4.16 the beam depletion for d(A)2 as a function of laser power is
shown. The collisional lifetime of d(A)2 is 5 s scaled from the lifetime of AMP (7 s)
and τ* is listed in table 2 (25 µs). Time t is estimated to be half a revolution time
of d(A)2. i.e., 49 µs. The uncertainty of eq.(32) comes from the estimation of time
t, i.e., how long is the time for the stored ions to travel to the other side of ELISA
and then give a signal in the detector. A time of 49 µs corresponds to traveling
from the middle of the straight section of ELISA to the middle of the other straight
section. So this time can vary by ? 10 µs which corresponds to traveling from one
end to the other end of the same straight section. This variation can change
eq.(32) by factor of 2.
From fig.4.16, the total depletion for d(A)2 is larger than that calculated
from eq.(32) indicating that there more than 90% of the ions decomposes via a
nonergodic process (factor of 2 uncertainty considered). This phenomenon was
also observed in the photodissociation of AMP cations (more than 99%
dissociation via nonergodic processes) while for AMP anions there was no
indication of nonergodic dissociation [173]. In the case of oligonucleotide
monocations, the protonation site is the nucleobase but the neutral bases may
also absorb UV light. We consider a simple model. If the protonated base
absorbs the photon then all ions dissociate via a nonergodic process while if the
neutral base absorbs the photon then all ions dissociate via an ergodic process.
Therefore the depletion of d(A)2 should be 50/50 via ergodic and nonergodic
processes if the photon absorption cross section is the same for both. The fact
that 90% ions decompose via a nonergodic process indicates that the absorption
cross section for a charged base is higher than that for a neutral base. If this is
true, increasing the number of neutral base can increase the photon-absorption
probability by the neutral base thus decrease the ratio between total depletion
and depletion from the ergodic process. In the case of d(A)5, there are four
neutral bases so the ratio decreases to 7 times while for d(A)2 it is 20 times
(fig.4.16).
Photodissociation of protonated amino acids

As discussed above, ions with well-defined initial energy will also have a well-
defined internal energy leading to an exponential decay in ELISA. But there are
exceptions. Here similar experiments as described above are done on
protonated tryptophan, but instead of an exponential decay, a power law decay is
75
_______________________________________________________
obtained (fig.4.17). As discussed in section 4.2, the power law decay indicates
that the internal energy distribution is broad. In the present experiments, TrpH+
was thermally equilibrated at room temperature and the average internal energy
E0 was 0.3 eV. The canonical internal energy distribution has a full width of 0.2
eV which is quite small compared to the photon energy (4.67 eV). Therefore
there must be an additional process involved. In the following we consider the
possible importance of fluorescence. Also the formation of the tryptophan radical
cation after prompt loss of hydrogen from TrpH+ is discussed since its decay
spectrum will be superimposed on that of TrpH+.
Fig.4.17 Decay spectrum of TrpH+ after 266-nm photon absorption. The

solid line is a fit to a 1/t decay curve including the backgrounds due to CID.
If the ms lifetime component of the TrpH+ decay is due to a decay from the
triplet state to the singlet ground state, there must be solvent quenching in water
solution where the triplet-state lifetime is of the order of microseconds. Another
possible process after photon absorption is fluorescence from the initially excited
Trp. In aqueous solution, the fluorescence quantum yield of Trp is larger than 0.1
and highly dependent on the pH of the solution (low when pH<4) [174]. Since
tryptophan has a higher fluorescence quantum yield than the other amino acids,
its fluorescence is often used to study the structure and dynamics of proteins in
solution [175]. The maximum light emission is around 350 nm and the internal
energy of TrpH+ is about 1.4 eV after fluorescence. The width of the internal
energy distribution includes the spread in the fluorescence energy, which is quite
large at room temperature ∼0.6 eV [176] which is more than sufficient to explain
a 1/t decay function. For example, with an activation energy of 1.2 eV and a pre-
exponential factor of 5? 1013 s−1 in the Arrhenius expression, the lifetime after
fluorescence covers the range from 100 µs to 0.1 s.
76
Lifetime measurement of radical ions
_______________________________________________________
However, very recent evidence by Kang et al. [177] suggests another
interpretation. The main dissociation channel of TrpH+ after 266-nm photon
absorption appears to be loss of a hydrogen to give the Trp radical cation. The
mechanism for this is believed to be a prompt direct process that involves an ion
where the proton is located on the indole ring and not on the amino nitrogen.
When the indole ring is protonated, fluorescence is strongly quenched. We have
calculated the energy required for loss of a hydrogen from protonated 3-
methylindole at the MP2/6-311++G(2d,p)//B3LYP/6-31+G(d) level to be 3.6 eV
(corrected for zero-point kinetic energies). The remaining average energy in the
Trp cations is then about 1 eV, and with an additional broadening of the
distribution associated with the hydrogen loss we are likely in the regime of 1/t
decay.
Summary
The width of the internal energy distribution of ions is highly important for the
observed decay of the ions. By using an ion trap we can obtain the ions with a
well-defined internal energy. The decay rate can be obtained by an exponential
fitting of the decay spectrum. If the final temperature is known, an Arrhenius plot
can be made and the activation energy and pre-exponential factor can be derived.
This is a complementary to BIRD measurements to determine the kinetic
parameters in the high temperature limit. In the case of the oligonucleotide
cations, most of the ions dissociate via a nonergodic processes. A deviation from
exponential decay is also observed for TrpH+ where instead a power law decay is
observed. A possible explanation could be due to fluorescence or loss of a
hydrogen from the indole ring.
4.4 Lifetime measurement of radical ions

As discussed in chapter 3, high energy collisions can produce radical ions. If we
inject the radical ions into ELISA, lifetimes of radical ions can be measured. In
order to produce radical ions a sodium oven is mounted after the magnet in the
injection section of ELISA.
Lifetime measurement of a charge-reduced peptide cation

We first produced the doubly protonated decapeptide Ser-Tyr-Ser-Met-Glu-His-
Phe-Arg-Trp-Gly (fragment 1–10 of ACTH (Adrenocorticotropic Hormone
Human)) by electrospray ionization. After 11-kV acceleration and selection of the
peptide dications by a magnet, these ions were collided with sodium vapour and
one-electron reduced ions, [M+2H]+·, of 22 keV translational energy were stored
in the storage ring and their lifetime measured (fig.4.18). Two components with
lifetimes of 0.3 ms and 1.8 ms are observed. Since the peptide is doubly charged
and electron capture can occur to both charge sites, two singly charged ions are
produced with different lifetimes. The exothermicity of the electron capture
reaction is ca 6 eV. Direct cleavage after electron attachment (in less than 1 ps)
is likely to be the major process (nonergodic fragmentation), but ions that decay
in this way do not contribute to the millisecond decay. On the assumption that
77
_______________________________________________________
capture occurs with equal probability to the two sites and that the branching
ratios between nonstatistical and statistical decays are the same, the areas
under the two decay curves should be equal. Indeed, this is the case for the fit in
fig. 4.18.
+
[M + 2H]
3
10 0.3 ms
Counts
1.8 ms
2
10
1
10
2 4 6 8 10 12 14 16 18 20
Time (ms)
Fig.4.18 Decay spectrum for a one-electron reduced decapeptide radical
cation. The data are fitted by two exponentials with the constraint that the
areas under the two curves should be equal.
Lifetime measurement of nucleotide dianions

Ions from an electrospray source were accumulated in an ion trap in which they
were thermalized by collisions with helium. The ion trap was kept at either room
temperature or −70 oC and emptied after nearly 0.1 sec. After acceleration to a
kinetic energy of 22 keV, nucleotide ions were selected with a bending magnet
and collided with sodium vapour. All ions in the bunch were injected into ELISA,
but the storage ring parameters were optimized for the storage of dianions
formed in the collisions. Monoanions, likely formed after electron autodetachment
from dianions in the ring, were measured in a channeltron detector located
opposite the injection side. The channeltron detector was positioned about 1 cm
from the circulating ion beam, and it was used since the dominant reaction
channel of the dianions is the production of monoanions. In another experiment,
the ions were dumped after 34 milliseconds of storage at the multichannel plate
(MCP) detector to identify any ions of long lifetime. The MCP is located at the
injection side (Fig. 2.3).
Decay spectra of the dianions formed after collisions with sodium vapour
are shown in Fig. 4.19 for two different initial temperatures of the AMP
monoanion (298 K and 203 K). There is no apparent temperature effect, and the
78
Lifetime measurement of radical ions
_______________________________________________________
2
298 K
10 203 K
Counts
1
10
0
τ = 0.2 ms
10
0.0 0.5 1.0 1.5 2.0

Time (ms)
Fig.4.19 Decay spectra of [AMP−H]2−. The spacing of the points is 74 µs, which is the
revolution time in the ring. The signal is measured in the channeltron detector.
90
60
Counts
30 40 µs
0
34.00 34.05 34.10 34.15
Time (ms)
Fig.4.20 The signal measured when the circulating [AMP−H]2− ion beam in ELISA is
dumped in the MCP detector after 34 ms of storage. Because of the poor resolution
of the magnet, the ion beam contains two isotope contributions (m/q 172.5 and m/q
173) which are separated in time by 40 µs (predicted separation 49 µs). Note, two
singly charged fragments of m/q 172 and 173 would separate in time by 99 µs.
79
_______________________________________________________
time constant is about 0.2 ms for both decays. This finding indicates that the
internal energy of the ions is determined by the electron capture and concomitant
hydrogen loss processes.
The count rate in the channeltron detector was too low to identify longer
lived components, and therefore any remaining ions were dumped directly in the
MCP detector after 34 milliseconds of storage. Indeed a signal is measured
(Fig.4.20), and a fraction of the ions therefore has a long lifetime of at least ten
milliseconds. At the B3LYP/6-311++G(2d,p)/PM3 level of theory we calculate the
energy for vertical attachment of an electron to [AMP−H] − (N-dehydrogenated
anion) to be slightly positive by 0.1 eV. Hence it is likely that the ions detected
after long time are stable to electron autodetachment.
80
Summary and outlook
________________________________________________________________
Chapter 5
Summary and outlook

The results of recent studies of biomolecular ions have been presented. Two
experimental setups were used in these studies: an accelerator mass
spectrometer and an electrostatic storage ring. The source for production of
biomolecular ions is electrospray. All ions are accelerated by a high voltage
acceleration tube and only interested ions are selected by a magnet. We have
focused on the differences from experiments at lower energies.
An aspect of high-energy collision experiments is the large cross section
for electron transfer between collision partners, either electron loss or capture to
the ion. Interesting radical ion chemistry can then be studied. For example,
collisional electron transfer between peptide multiply charged cations and sodium
gas generates radical ions that possess an additional electron. These radical
ions display characteristic fragmentation patterns that are governed by their
radical chemistry, Coulomb repulsion between charges, and/or fast nonstatistical
processes. Electron detachment from oligonucleotide anions also promotes a
selective enhancement of certain fragmentation processes. Exotic dianions can
also be produced by high energy collisions between anions and sodium vapour.
In the case of nucleotides, the excess electron is captured by the nucleobase
inducing hydrogen loss from a N−H group.
At the electrostatic storage ring we have studied the time dependence of
the decay of large molecular ions. As an example, we have studied the power-
law decay of collisionally activated amino acids and oligonucleotides and
determined the quenching time for radiative cooling. The decay of nucleotide ions
after photoexcitation has been analyzed to give information on the relative
importance of statistical and nonstatistical fragmentation processes. In
photoexcitation experiments the energy is initially located in the chromophore
part of the molecule and then spreads to the rest of the molecule within about a
picosecond. Fragmentation after intramolecular vibrational redistribution (IVR) is
a statistical process whereas dissociation before IVR or from electronic excited
states is nonstatistical. Only decay on a time scale of micro- to milliseconds can
be measured directly in the ring, but prompt (nonstatistical) decay is revealed as
a depletion of the beam after photoexcitation.
In another experiment, we have taken advantage of the high cross section
for electron capture in high-energy collisions with sodium vapour to produce
radical ions, and we have measured the time dependence of their fragmentation
in the storage ring. In the case of charge-reduced peptide ions, we identify two
different species formed upon electron attachment to a peptide dication. Both of
the two charged ends of the peptide can equally well capture an electron. In the
measurement of AMP dianions, a lifetime of 0.2 ms is measured independent of
the initial internal energy of the ions indicating that the internal energy of the ions
is determined by the electron capture and concomitant hydrogen loss processes.
81
Summary and outlook
________________________________________________________________
Another electrostatic storage ring has been built recently in Tokyo [178],
which is similar to ELISA with an electron target for merged beam experiments
Also, Stockholm University has a project to build a double electrostatic storage
ring named Double Electrostatic Storage Ion Ring ExpEriment (DESIREE).
Positive and negative ions can be merged in the common straight section of the
ring. Their interesting reactions can be studied.
In future work collisions between multiply charged oligonucleotide anions
and sodium are planned to study damage to nucleic acids by electron capture.
Similar experiments can also be done on duplex oligonucleotides to reveal what
the excess electron does to the duplex structure.
Future work on the photoexcitation of amino acids, peptides and
oligonucleotides is planned with the purpose of better understanding the
photophysics of these molecules. We are especially interested in identifying
‘prompt’ nonstatistical processes on the picosecond timescale. Another theme
will be the transition from isolated to solvated molecular ions. Stepwise solvation
of the ions will be carried out to bridge the gap between gas phase and solution
phase.
82
Appendix
_______________________________________________________
Appendix A
In-source activation between tube lens and skimmer

As discussed in chapter 2, in-source collisional activation sometimes can change
the fragmentation pattern especially for fragile primary ions. As shown in fig.2.2
the ions are accelerated between the tube lens and skimmer. During the passage
the internal energy of the ions can be increased if the ions collide with the
residual gas. This energy is enough to destroy the fragile ions. For example,
putting a higher voltage on the tube lens can make the average charge state of
the ions selected by the mass spectrometer shift to a lower value [45,46]. Here I
present an example to demonstrate the importance of the tube lens voltage.
Previously, collisions between 50-keV fluorinated fullerenes (C60Fx-) and
noble gas (Xe) resulted in observation of dianions of highly fluorinated fullerenes.
Application of the plasma ion source and ESI source, afforded “hot” and “cold”
fluorofullerene anion beams, respectively. In the latter case, non-dissociative
electron capture process prevailed over a dissociative process, for which the
cross sections measurements were performed [179].
Now instead of Xe gas we used sodium as a target. In addition to the
dianions, trianions are observed in the MIKE spectrum (fig.1). One-fluorine-loss
dianions are also observed but not dominant over the intact dianions when the
tube lens potential is low. However if we apply a high tube lens voltage the
fragment dianions have higher yields than the intact ones. In fig.2 the ratio of
fragment dianions and intact dianions (γ) is shown as a function of tube lens
voltage. Thus the ratio γ can be considered as a thermometer to measure the
temperature of the primary ions. In fig.3 the relative formation cross section for
the trianion is plotted as a function of γ. The cross section strongly depends on
the temperature (γ) of the primary ions. At certain temperature the trianions can
even be missing in the spectrum indicating the electron autodetachment is much
faster than 10 µs.
The electron autodetachment can be interpreted as tunnelling through the
Coulomb barrier mainly from a thermally populated excited electronic state or
with transfer of energy to the electron from hot vibrational bands. This
phenomenon is discussed in details in the study of decay of free C60 dianions
[180].
83
Appendix
_______________________________________________________
50000 − o −
C60F47+Na (190 C) C60F47
2−
C60F47
40000
30000
Counts
20000
3-
10000 C60F47
×10
0
0 200 400 600 800 1000 1200 1400 1600
m/q
Fig. 1 MIKE spectrum obtained after collisions of C60F47− with Na
Note, that the intensity of the peak at 537.7 is multiplied by 10.
1.4
1.2
1.0
I(C60F46 )/I(C60F47 )
2-
0.8
0.6
2-
0.4
0.2
0.0
60 80 100 120 140 160 180 200 220
Tube lens voltage (V)
Fig. 2 Ratio between fragment dianions and intact dianions as a function of
tube lens potential. Note with a high tube lens voltage the ratio is larger than 1.
84
Appendix
_______________________________________________________
14
12
10
8
σrel
0
0.0 0.2 0.4 0.6 0.8 1.0 1.2
γ
Fig. 3 Relative formation cross section of trianions as a function of γ.
85
Appendix
_______________________________________________________
Appendix B
Biomolecular cluster ions

There is an interest in clusters of biomolecules [31-33, 181,182]. For example,
Cooks and coworkers found that the serine octomer had unusual stability in the
gas phase and moreover L-and D-serine can recognize each other to form the
stable cluster form [31-33]. We also performed the experiments on serine
clusters shown in fig.1. It is obvious that the octomer is the most intense among
all the clusters. In fig. 2 the chiral recognition is observed. For the mixture of L-
and D-serine the peak of the octomer is comparable with the others. But in the
case of D-serine, the octomer peak is dominant over other clusters.
A sonic spray ion source is planned to be used for further studies of
biomolecular clusters.
+
8000 [Ser8+H]
3500 +
[Ser9+H]
3000
+
2500 [Ser10+H]
Counts
6000 + 2000 2+
[Ser5+H] [Ser19+H]
1500
4+
1000
[Ser39+H]
+
[Ser6+H]
Counts
500
4000 0
945 997.5 1050
+
[Ser7+H]
2000
0
600 800 1000 1200 1400 1600
m/q
Fig. 1 Magnet scan of ions formed from ESI. In addition to single-charged
serine clusters, multiply charged serine clusters are also observed. Serine
octomer is most intense.
86
Appendix
_______________________________________________________
Mixture of L and D
+ +
[Ser5+H] [Ser6+H]
+
[Ser8+H]
+ +
[Ser7+H] [Ser9+H]
550 600 650 700 750 800 850 900 950

D only
550 600 650 700 750 800 850 900 950

m/q
Fig. 2 Magnet scan of mixture of L- and D-serine and D-serine
only. The octomer is not dominant in mixture of L- and D- serine,
while for only D-serine the octomer again becomes most intense.
87
Appendix
_______________________________________________________
Appendix C
Calculating the microcanonical caloric curve
The vibrational frequencies can be calculated from the Gaussian 98 program.
Then the canonical caloric curve is given by
1
E(T) = ∑ Khw i (1)
hw i
i
exp(K ) −1
k BT
where K is 1.24×10-4, the conversion factor for energy from cm-1 to eV. The last
term is eq.(1) is the average number of oscillators at temperature T with
frequency ω.
So the derivative with respect to temperature T gives the canonical heat capacity
as a function of T (eq.(2)).
hw i 2 hw i 1
C(T) = ∑ (K ) exp(K )( ) 2 (2)
k BT k BT hw
i
exp(K i
) −1
k BT
Then the microcanonical caloric curve is calculated given by eq.(3)
E m (T) = E(T) − k BT
(3)
C m (T) = C(T) − k B
88
Appendix
_______________________________________________________
Appendix D
Calculating the radiation power from hot molecules
The radiative power can be calculated according to eq.(26) in chapter 4.
2ω 2 q 2 hω
Pr = (1)
3Mc exp( hω/k BT) − 1
3
From the Gaussian calculations not only frequencies are calculated but also IR
Intensities (q2/M) which is in units of km/mol. What we need for radiative power
is eV/s, so the unit conversion is carried out in the following:
IR intensity divided by 42.255 gives unit of D2/Å2⋅amu. 1 D2/Å2⋅amu is equal to
1
2
e2/amu. So eq.(1) now becomes
4.803
IR 2 ω2e2 hω
Pr = where IR stands for IR intensity.
42.255 ⋅ 4.803 3 amu ⋅ c exp( hω/k BT) − 1
2 3
e2 is equal to 14.4 eV⋅Å while 1 amu⋅c2 is equal to 931.5×106 eV, so the upper
equation can be simplied by
IR 2 14.4 ω2 1
Pr = (Å) < n > hω with < n >=
42.255 ⋅ 4.803 3 931.5 ×10
2 6
c exp( hω/k BT) − 1
Multiplying 1.24×10 to hω converts from cm to eV. The frequency given by
4 -1
Gaussian is not the circular frequency so we have to multiply frequencies ( f

given by Gaussian) by 2π. We have to multiply the speed of light c to the
ω2
frequency to convert from cm-1 to s-1. Concerning Å in the equation, gives a
c
factor of 4π2⋅300. So, finally the radiative power is given by
1 2 14.4
Pr = 4π 2 ⋅ 300 ⋅1.24 ×10 −4 < n > f 3 ⋅ IR (eV/s)
42.255 ⋅ 4.803 3 931.5 × 10 6
2
89
Appendix
_______________________________________________________
Appendix E
Absorption rate from blackbody radiation
When we cool down the ions in the ion trap, ions are in equilibrium with the trap.
After injection to ELISA, the ions can absorb photons emitted from the walls of
ELISA. This absorption heats up the molecules from the original temperature of
the trap. It is necessary to calculate the absorption rate.
Absorption rate R is given by

8 πhυ 3 µ2
R= < n > (1)
c3 6 ε0 h 2
hω
where µ is the transition dipole moment. In Gaussian calculation µ 2 = IR
ω2
so insert into eq.(1)
8πυ 2 IR
R= 3 <n> (2) (in SI unit)
c 6ε 0
In Gaussian unit, eq.(2) becomes
32 π 2 υ 2 IR
R= 3
<n>
c 6
By applying the same procedure in Appendix C
Finally absorption rate R is given by
32π 2 1 14.4
R= 300 < n > ⋅f 2 ⋅ IR (s-1)
6 42.255 ⋅ 4.803 931.5 × 10 6
2
90
Reference
_____________________________________________________________
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PhD Thesis

Biomolecular Ions in Accelerators

Department of physics and Astronomy

3 Biomolecular ions in an accelerator mass

4 Biomolecular ions in ELISA

5 Summary and outlook 81

Fig.1.1 Nucleobases in (a) DNA and (b) RNA.

2’ carbon. A sugar and a nucleobase can be combined by a glycosidic linkage to form a

Fig.1.4 Adenosine 5’-monophosphate (AMP).

Fig.1.6 Polymerization of (a) nucleotides and (b) amino acids.

From brick to mansion

Fig.1.7 (a) DNA double helix (b) RNA hairpin loop.

Thymine Adenine Cytosine Guanine

Fig.1.8 Watson-Crick base paring.

1.2 Soft ionization techniques

Fig.1.10 Formation of Taylor cone at the needle.

Fig.1.11 Scheme of formation of small charged droplets.

Fig.1.12 Electrospray ion mass (m/q) spectra

Soft Laser desorption

called matrix-assisted laser-desorption ionization (MALDI) [27,28]. Nowadays, the

Fig.1.13 Scheme of process of MALDI.

Fig.1.14 Sonic spray ionization source from reference [31].

Sonic spray ionization

Fig.1.15 Research on biomolecules in the gas phase.

1.3 Study of biomolecules in the gas phase

2.1 Accelerator mass spectrometer

gas collision cell

Fig.2.1 Schematic drawing of accelerator mass spectrometer.

1 mbar 10-3 mbar 10-5 mbar 10-6 mbar

Rotary Turbo Turbo

Fig.2.2 Schematic drawing of electrospray ion source.

Fig.2.3 Schematic drawing of the electrostatic storage ring (ELISA).

Typical timing sequence for ELISA

ELISA close timing

Fig.2.4 Typical timing sequence for ELISA.

The 22-pole ion trap

moves. The amplitude of micromotion is proportional to the instantaneous amplitude of the

Fig.2.6 Combination of secular motion and micromotion.

Fig.2.7 Illustration of 22-pole ion trap.

Table 1 number of ions in a bunch (calibration based on [54]).

Fig.2.8 (a) Pickup signals for AMP anions stored in

2.3 Sample preparation

Biomolecular ions in an accelerator mass

Substituting (3) into (2), we have

Fig.3.1 Schematic drawing of the collision process. The intensity of the

3.2 Multiply-charged oligonucleotide anions in

0 200 400 600 800 1000 1200 1400 1600

Single and double electron loss from oligonucleotides

Fig.3.4 Single electron loss cross section for [dA5-3H] 3- in He.

Fig.3.5 Electron loss cross section of dA7 as a function of charge state.

Electron-loss induced dissociation

0 200 400 600 800 1000 1200 1400 1600

0 200 400 600 800 1000 1200 1400

In the beginning of the 90’s, low-energy CID experiments on oligonucleotide anions

Scheme 3.1 Nomenclature for fragmentation of oligonucleotides.

A mechanism for production of a ions based on single-electron loss is proposed in

Scheme 3.3 The proposed mechanism for production of a ions.

0 200 400 600 800 1000 1200 1400 1600