Papers by Francesco Lanucara
International journal of mass spectrometry
Rapid Communications in Mass Spectrometry
Chemical Physical Letters
A stable N1-nitroso derivative of N-acetyltryptophan, [NANT-H], has been assayed by infrared mul... more A stable N1-nitroso derivative of N-acetyltryptophan, [NANT-H], has been assayed by infrared multiple photon dissociation (IRMPD) in the ‘fingerprint’ range. IRMPD spectra, interpreted by DFT calculations, display features characteristic of the nitrosation motif, which lack in the native N-acetyltryptophan anion, [NAT-H]. The most stable [NANT-H] isomers, nicely accounting for the experimental features, present the carboxylic group interacting with the amide and benzene ring hydrogens. The side chain is orientedgauche with respect to the indole plane, while the NNO group may adopt either a syn (1ds, global minimum)or an anti (2da, 2.7 kJ mol1 higher in energy) configuration.
Journal of the American Society for Mass Spectrometry
A one-step enzymatic reaction for improving the collision-induced dissociation (CID)-based tandem... more A one-step enzymatic reaction for improving the collision-induced dissociation (CID)-based tandem mass spectrometry (MS/MS) analysis of phosphorylated peptides in an ion trap is presented. Carboxypeptidase-B (CBP-B) was used to selectively remove C-terminal arginine or lysine residues from phosphorylated tryptic/Lys-C peptides prior to their MS/MS analysis by CID with a Paul-type ion trap. Removal of this basic C-terminal residue served to limit the extent of gas-phase neutral loss of phosphoric acid (H 3 PO 4 ), favoring the formation of diagnostic b and y ions as determined by an increase in both the number and relative intensities of the sequence-specific product ions. Such differential fragmentation is particularly valuable when the H 3 PO 4 elimination is so predominant that localizing the phosphorylation site on the peptide sequence is hindered. Improvement in the quality of tandem mass spectral data generated by CID upon CBP-B treatment resulted in greater confidence both in assignment of the phosphopeptide primary sequence and for pinpointing the site of phosphorylation. Higher Mascot ion scores were also generated, combined with lower expectation values and higher delta scores for improved confidence in site assignment; Ascore values also improved. These results are rationalized in accordance with the accepted mechanisms for the elimination of H 3 PO 4 upon low energy CID and insights into the factors dictating the observed dissociation pathways are presented. We anticipate this approach will be of utility in the MS analysis of phosphorylated peptides, especially when alternative electron-driven fragmentation techniques are not available.
Biochemical Society Transactions, 2013
The Journal of Physical Chemistry Letters
S-nitrosation of cysteine plays an important role in storage and transport of NO, a key signaling... more S-nitrosation of cysteine plays an important role in storage and transport of NO, a key signaling molecule in vivo. An approach to detect this modification in the bare, charged amino acid is presented, based on IR multiple photon dissociation (IRMPD) spectroscopy. Protonated and deprotonated S-nitrosocysteine ions, [SNOCys+H]+ and [SNOCys−H]−, have been obtained by electrospray ionization and assayed for IR activity in either the 1000–1900 cm−1 or the 3000–3600 cm−1 wavenumber range. The so-obtained IRMPD spectra display characteristic features ascribed to the presence of the S-nitrosation motif, which are missing in the corresponding IRMPD spectra of the native cysteine ions, [Cys+H]+ and [Cys−H]−. In particular, the NO stretching mode is unambiguously identified by the red shift observed for the 15N-labelled species. The interpretation of the IRMPD spectra is supported by density functional theory calculations of the optimized geometries, relative energies and IR spectra of [SNOCys+H]+ and [SNOCys−H]−. Both sampled ions comprise a thermally averaged population of conformers contributing to the experimental IRMPD spectra. This notion is supported by the agreement between the calculated IR absorption spectra of the several conformers, and the recorded IRMPD spectrum. The gathered evidence points to a characteristic NO stretching mode that emerges as a pronounced feature at 1460–1490 cm−1 in the IRMPD spectrum of [SNOCys−H]−, namely in a region where [Cys−H]− displays no IRMPD activity. Conversely, the NO stretching vibration of [SNOCys+H]+ is enclosed in a wide absorption including the C► Protonated and deprotonated S-nitrosocysteine ions are assayed in the gas phase by IRMPD spectroscopy. ► The ions from S-nitrosocysteine are examined in parallel with the ions from the native amino acid. ► The spectrum of the anion (red) reveals a νNO feature in a blank portion of the spectrum of deprotonated cysteine (green). ► DFT calculations underline the presence of several conformers contributing to the overall IR spectrum (black).
Methods in Enzymology, 2011
One of the main goals of comparative cell signaling analyses is the characterization of protein c... more One of the main goals of comparative cell signaling analyses is the characterization of protein changes between different biological samples, either globally or by targeting specific proteins of interest. Highly accurate and precise strategies are thus required for the relative quantification of proteins extracted from two or more different cell populations. Stable isotope labeling with amino acids in cell culture (SILAC) is a general method for mass spectrometric quantitative proteomics based on metabolic incorporation of stable isotope-labeled amino acids into the cellular protein pool. This method has been applied with great success to a variety of quantitative proteomics problems aimed at gaining further insight into cell signaling pathways. In this chapter, we describe how SILAC can be used for the elucidation of cellular mechanisms, including temporal proteome profiling and the quantitative analysis of the extent of specific posttranslational modifications.
Methods in Enzymology, 2011
Building a mathematical model of a biological system requires input of experimental data for each... more Building a mathematical model of a biological system requires input of experimental data for each networked component, ultimately generating a model that can be used to test scientific hypotheses. A fundamental requirement in the computation of these systems is that the total amount of each component can be specified precisely. An added level of complexity occurs because a vast number of protein posttranslational modifications modulate protein function. Each of these modified forms therefore needs to be considered as a separate system component, and must therefore be quantified individually. In this chapter, we describe how designer QconCAT proteins can be used to determine the absolute amounts of both the polypeptide components and their covalently modified derivatives in both yeast and mammalian extracts derived from living cell populations.
S-nitrosation of cysteine plays an important role in storage and transport of NO, a key signaling... more S-nitrosation of cysteine plays an important role in storage and transport of NO, a key signaling molecule in vivo. An approach to detect this modification in the bare, charged amino acid is presented, based on IR multiple photon dissociation (IRMPD) spectroscopy. Protonated and deprotonated S-nitrosocysteine ions, [SNOCys+H]+ and [SNOCys-H]-, have been obtained by electrospray ionization and assayed for IR activity in either the 1000-1900 cm-1 or the 3000-3600 cm-1 wavenumber range. The so-obtained IRMPD spectra display characteristic features ascribed to the presence of the S-nitrosation motif, which are missing in the corresponding IRMPD spectra of the native cysteine ions, [Cys+H]+ and [Cys-H]-. In particular, the NO stretching mode is unambiguously identified by the red shift observed for the 15N-labelled species. The interpretation of the IRMPD spectra is supported by density functional theory calculations of the optimized geometries, relative energies and IR spectra of [SNOCys+H]+ and [SNOCys-H]-. Both sampled ions comprise a thermally averaged population of conformers contributing to the experimental IRMPD spectrum. This notion is supported by the agreement between the convoluted IR spectrum of the several conformers, and the recorded IRMPD spectrum. The gathered evidence points to a characteristic NO stretching mode that emerges as a pronounced feature at 1460-1490 cm-1 in the IRMPD spectrum of [SNOCys-H]-, namely in a region where [Cys-H]- displays no IRMPD activity. Conversely, the NO stretching vibration of [SNOCys+H]+ is enclosed in a wide absorption including the C=O stretching mode at 1780 cm-1. The [SNOCys-H]- negative ions are thus a promising benchmark in a search for S-nitrosation features using IRMPD spectroscopy.
Protein post-translational modifications (PTMs) are critically important in regulating both prote... more Protein post-translational modifications (PTMs) are critically important in regulating both protein structure and function, often in a rapid and reversible manner. Due to its sensitivity and vast applicability, mass spectrometry (MS) has become the technique of choice for analyzing PTMs. Whilst the ''bottom-up' analytical approach, in which proteins are proteolyzed generating peptides for analysis by MS, is routinely applied and offers some advantages in terms of ease of analysis and lower limit of detection, ''top-down'' MS, describing the analysis of intact proteins, yields unique and highly valuable information on the connectivity and therefore combinatorial effect of multiple PTMs in the same polypeptide chain. In this review, the state of the art in top-down MS will be discussed, covering the main instrumental platforms and ion activation techniques. Moreover, the way that this approach can be used to gain insights on the combinatorial effect of multiple post-translational modifications and how this information can assist in studying physiologically relevant systems at the molecular level will also be addressed. # 2012 Wiley Periodicals, Inc. Mass Spec Rev
The intrinsic reactivity of a
manganese(V)-oxo porphyrin complex,
a typically fleeting intermedia... more The intrinsic reactivity of a
manganese(V)-oxo porphyrin complex,
a typically fleeting intermediate in catalytic
oxidation reactions in solution,
has been elucidated in a study focused
on its gas-phase ion-chemistry. The
naked high-valent MnV-oxo porphyrin
intermediate 1 ([(tpfpp)MnVO]+;
tpfpp=meso-tetrakis(pentafluorophenyl)
porphinato dianion), has been obtained
by controlled treatment of
[(tpfpp)MnIIICl]&&square brackets
have been used throughout also for the
neutral species, ok?&& (2-Cl) with iodosylbenzene
in methanol, delivered&
&“generated”, better?&& in the gas
phase by electrospray ionization and
assayed by FT-ICR mass spectrometry.
A direct kinetic study of the reaction
with selected substrates, each containing
a heteroatom X (X=S, N, P) including
amines, sulfides, and phosphites,
was thus performed. Ionic products
arising from electron transfer
(ET), hydride transfer (HT), oxygenatom
transfer (OAT), and formal addition
(Add) may be observed, with a
predominance of two-electron processes,
whereas the product of hydrogenatom
transfer (HAT),
[(tpfpp)MnIVOH]+, is never detected.
A thermochemical threshold for the
formation of the product radical cation
allows an evaluation of the electrontransfer
ability of a MnV-oxo complex,
yielding a lower limit of 7.85 eV for the
ionization energy of gaseous
[(tpfpp)MnIVO]. Linear free-energy
analyses of the reactions of para-substituted
N,N-dimethylanilines and thioanisoles
indicate that a considerable
amount of positive charge is developed
on the heteroatom in the oxidation
transition state. Substrates endowed
with different heteroatoms, but similar
ionization energy display a comparable
reaction efficiency, consistent with a
mechanism initiated by ET. For the
first time, the kinetic acidity of putative
hydroxo intermediates playing a role in
catalytic oxidations, [(tpfpp)FeIVOH]+
and [(tpfpp)MnIVOH]+, has been investigated
with selected reference
bases, revealing a comparatively higher
basicity for the ferryl, [(tpfpp)FeIVO],
with respect to the manganyl,
[(tpfpp)MnIVO], unit. Finally, the neat
association reaction of 2 has been studied
with various ligands showing that
harder ligands are more strongly
bound.
"Functional models of the Compound I intermediate of monooxygenase heme enzymes, namely [(TPFPP)•... more "Functional models of the Compound I intermediate of monooxygenase heme enzymes, namely [(TPFPP)•+FeIV=O]+ and [(TPFPP)MnV=O]+ (TPFPP = meso-tetrakis (pentafluorophenyl)porphyrinato dianion), are obtained as bare species by electrospray ionization from solutions of appropriate precursors and their reactivity is investigated in the gas-phase. By an alternative approach involving the reaction of a gaseous oxidant, the naked core of Compound I, [(PP-IX)•+FeIV=O]+ (PP-IX = protoporphyrin IX dianion) has been produced as well. This achievement, unprecedented in studies run in solution, is now made possible working in the gas-phase. The long lifetime ensured by the dilute gas-phase allows to reveal both structural details and elementary steps of the catalytic activity of these high-valent oxo-metal intermediates. Depending on the features of the oxo-metal complex, ionic products are formed with neutral substrates involving: (i) addition, (ii) oxygen atom transfer, (iii) formal hydride transfer. In contrast, ionic products indicative of a net initial hydrogen atom transfer event are never observed. The reaction pathways of these ultimate catalytic intermediates void of any trans axial ligand, counterion, solvent or protein environment are thus elucidated.
"
S-Nitrosocaptopril, a biologically active S-nitrosothiol, is generated as protonated species and ... more S-Nitrosocaptopril, a biologically active S-nitrosothiol, is generated as protonated species and isolated in the gas phase by electrospray ionization coupled to Fourier Transform Ion Cyclotron Resonance (FT-ICR) or ion-trap mass spectrometry. The structural and IR spectroscopic characterization of protonated S-nitrosocaptopril (SNOcapH(+)) is aided by the comparative study of the parent species lacking the NO feature, namely protonated captopril. The study is accomplished by methodologies based on tandem mass spectrometry, namely by energy resolved collision-induced dissociation and infrared multiple-photon dissociation (IRMPD) spectroscopy, backed by density functional theory calculations. IRMPD spectra have been obtained both in the 1000-1900 cm(-1) fingerprint range, using a beamline of the infrared free electron laser (IR-FEL) at the Centre Laser Infrarouge d'Orsay (CLIO), and in the O-H and N-H stretching region (2900-3700 cm(-1)) using the tunable IR radiation of a tabletop parametric oscillator/amplifier (OPO/OPA) laser source. The structural features of the ion have been ascertained by comparison of the experimental IRMPD spectra with the IR transitions calculated for the lowest energy isomers. Evidence is obtained that protonation occurs at the amide carbonyl oxygen which is found to be the thermodynamically most basic site. However, SNOcapH(+) is present as a thermally equilibrated mixture of low-energy structures, with a major contribution of the most stable isomer characterized by a trans relationship of the positively charged OH group with respect to the carboxylic acid functionality on the adjacent proline ring and by an anti conformation at the S-N (partial) double bond, though the energy difference with the analogous trans-syn isomer is less than 1 kJ mol(-1). The highly diagnostic N-O stretching mode has been unambiguously identified, which may be regarded as an informative probe for S-nitrosation features in more complex, biologically active molecules.
Model ferric heme nitrosyl complexes, [Fe(TPP)(NO)]+ and [Fe(TPFPP)(NO)]+, where TPP is the diani... more Model ferric heme nitrosyl complexes, [Fe(TPP)(NO)]+ and [Fe(TPFPP)(NO)]+, where TPP is the dianion of 5,10,15,20-tetrakis-phenyl-porphyrin and TPFPP is the dianion of 5,10,15,20-tetrakis-pentafluorophenyl-porphyrin, have been obtained as isolated species by the gas phase reaction of NO with [FeIII(TPP)]+ and [FeIII (TPFPP)]+ ions delivered in the gas phase by electrospray ionization, respectively. The so-formed nitrosyl complexes have been characterized by vibrational spectroscopy also exploiting 15N-isotope substitution in the NO ligand. The characteristic NO stretching frequency is observed at 1825 and 1859 cm−1 for [FeIII(TPP)(NO)]+ and [FeIII(TPFPP)(NO)]+ ions, respectively, providing reference values for genuine five-coordinate FeIII(NO) porphyrin complexes differing only for the presence of either phenyl or pentafluorophenyl substituents on the meso positions of the porphyrin ligand. The vibrational assignment is aided by hybrid density functional theory (DFT) calculations of geometry and electronic structure and frequency analysis which clearly support a singlet spin electronic state for both [Fe(TPP)(NO)]+ and [Fe(TPFPP)(NO)]+ complexes. Both TD-DFT and CASSCF calculations suggest that the singlet ground state is best described as FeII(NO+) and that the open-shell AFC bonding scheme contribute for a high-energy excited state. The kinetics of the NO addition reaction in the gas phase are faster for [FeIII(TPFPP)]+ ions by a relatively small factor, though highly reliable because of a direct comparative evaluation. The study was aimed at gaining vibrational and reactivity data on five-coordinate FeIII(NO) porphyrin complexes, typically transient species in solution, ultimately to provide insights into the nature of the Fe(NO) interaction in heme proteins.
PROTEOMICS- …, Jan 1, 2010
Keywords:
* Autophagy;
* Degradative proteomics;
* Disease;
* Proteasome;
* ... more Keywords:
* Autophagy;
* Degradative proteomics;
* Disease;
* Proteasome;
* Ubiquitin
Abstract
Protein degradation is a fundamental biological process, which is essential for the maintenance and regulation of normal cellular function. In humans and animals, proteins can be degraded by a number of mechanisms: the ubiquitin-proteasome system, autophagy and intracellular proteases. The advances in contemporary protein analysis means that proteomics is increasingly being used to explore these key pathways and as a means of monitoring protein degradation. The dysfunction of protein degradative pathways has been associated with the development of a number of important diseases including cancer, muscle wasting disorders and neurodegenerative diseases. This review will focus on the role of proteomics to study cellular degradative processes and how these strategies are being applied to understand the molecular basis of diseases arising from disturbances in protein degradation.
J. Am. Chem. …, Jan 1, 2008
The mechanisms of oxidative N-dealkylation of amines by heme enzymes including peroxidases and cy... more The mechanisms of oxidative N-dealkylation of amines by heme enzymes including peroxidases and cytochromes P450 and by functional models for the active Compound I species have long been studied. A debated issue has concerned in particular the character of the primary step initiating the oxidation sequence, either a hydrogen atom transfer (HAT) or an electron transfer (ET) event, facing problems such as the possible contribution of multiple oxidants and complex environmental effects. In the present study, an oxo iron(IV) porphyrin radical cation intermediate 1, [(TPFPP)*+ Fe(IV)=O]+ (TPFPP = meso-tetrakis (pentafluorophenyl)porphinato dianion), functional model of Compound I, has been produced as a bare species. The gas-phase reaction with amines (A) studied by ESI-FT-ICR mass spectrometry has revealed for the first time the elementary steps and the ionic intermediates involved in the oxidative activation. Ionic products are formed involving ET (A*+, the amine radical cation), formal hydride transfer (HT) from the amine ([A(-H)]+, an iminium ion), and oxygen atom transfer (OAT) to the amine (A(O), likely a carbinolamine product), whereas an ionic product involving a net initial HAT event is never observed. The reaction appears to be initiated by an ET event for the majority of the tested amines which included tertiary aliphatic and aromatic amines as well as a cyclic and a secondary amine. For a series of N,N-dimethylanilines the reaction efficiency for the ET activated pathways was found to correlate with the ionization energy of the amine. A stepwise pathway accounts for the C-H bond activation resulting in the formal HT product, namely a primary ET process forming A*+, which is deprotonated at the alpha-C-H bond forming an N-methyl-N-arylaminomethyl radical, A(-H)*, readily oxidized to the iminium ion, [A(-H)]+. The kinetic isotope effect (KIE) for proton transfer (PT) increases as the acidity of the amine radical cation increases and the PT reaction to the base, the ferryl group of (TPFPP)Fe(IV)=O, approaches thermoneutrality. The ET reaction displayed by 1 with gaseous N,N-dimethylaniline finds a counterpart in the ET reactivity of FeO+, reportedly a potent oxidant in the gas phase, and with the barrierless ET process for a model (P)*+ Fe(IV)=O species (where P is the porphine dianion) as found by theoretical calculations. Finally, the remarkable OAT reactivity of 1 with C6F5N(CH3)2 may hint to a mechanism along a route of diverse spin multiplicity.
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Papers by Francesco Lanucara
manganese(V)-oxo porphyrin complex,
a typically fleeting intermediate in catalytic
oxidation reactions in solution,
has been elucidated in a study focused
on its gas-phase ion-chemistry. The
naked high-valent MnV-oxo porphyrin
intermediate 1 ([(tpfpp)MnVO]+;
tpfpp=meso-tetrakis(pentafluorophenyl)
porphinato dianion), has been obtained
by controlled treatment of
[(tpfpp)MnIIICl]&&square brackets
have been used throughout also for the
neutral species, ok?&& (2-Cl) with iodosylbenzene
in methanol, delivered&
&“generated”, better?&& in the gas
phase by electrospray ionization and
assayed by FT-ICR mass spectrometry.
A direct kinetic study of the reaction
with selected substrates, each containing
a heteroatom X (X=S, N, P) including
amines, sulfides, and phosphites,
was thus performed. Ionic products
arising from electron transfer
(ET), hydride transfer (HT), oxygenatom
transfer (OAT), and formal addition
(Add) may be observed, with a
predominance of two-electron processes,
whereas the product of hydrogenatom
transfer (HAT),
[(tpfpp)MnIVOH]+, is never detected.
A thermochemical threshold for the
formation of the product radical cation
allows an evaluation of the electrontransfer
ability of a MnV-oxo complex,
yielding a lower limit of 7.85 eV for the
ionization energy of gaseous
[(tpfpp)MnIVO]. Linear free-energy
analyses of the reactions of para-substituted
N,N-dimethylanilines and thioanisoles
indicate that a considerable
amount of positive charge is developed
on the heteroatom in the oxidation
transition state. Substrates endowed
with different heteroatoms, but similar
ionization energy display a comparable
reaction efficiency, consistent with a
mechanism initiated by ET. For the
first time, the kinetic acidity of putative
hydroxo intermediates playing a role in
catalytic oxidations, [(tpfpp)FeIVOH]+
and [(tpfpp)MnIVOH]+, has been investigated
with selected reference
bases, revealing a comparatively higher
basicity for the ferryl, [(tpfpp)FeIVO],
with respect to the manganyl,
[(tpfpp)MnIVO], unit. Finally, the neat
association reaction of 2 has been studied
with various ligands showing that
harder ligands are more strongly
bound.
"
* Autophagy;
* Degradative proteomics;
* Disease;
* Proteasome;
* Ubiquitin
Abstract
Protein degradation is a fundamental biological process, which is essential for the maintenance and regulation of normal cellular function. In humans and animals, proteins can be degraded by a number of mechanisms: the ubiquitin-proteasome system, autophagy and intracellular proteases. The advances in contemporary protein analysis means that proteomics is increasingly being used to explore these key pathways and as a means of monitoring protein degradation. The dysfunction of protein degradative pathways has been associated with the development of a number of important diseases including cancer, muscle wasting disorders and neurodegenerative diseases. This review will focus on the role of proteomics to study cellular degradative processes and how these strategies are being applied to understand the molecular basis of diseases arising from disturbances in protein degradation.
manganese(V)-oxo porphyrin complex,
a typically fleeting intermediate in catalytic
oxidation reactions in solution,
has been elucidated in a study focused
on its gas-phase ion-chemistry. The
naked high-valent MnV-oxo porphyrin
intermediate 1 ([(tpfpp)MnVO]+;
tpfpp=meso-tetrakis(pentafluorophenyl)
porphinato dianion), has been obtained
by controlled treatment of
[(tpfpp)MnIIICl]&&square brackets
have been used throughout also for the
neutral species, ok?&& (2-Cl) with iodosylbenzene
in methanol, delivered&
&“generated”, better?&& in the gas
phase by electrospray ionization and
assayed by FT-ICR mass spectrometry.
A direct kinetic study of the reaction
with selected substrates, each containing
a heteroatom X (X=S, N, P) including
amines, sulfides, and phosphites,
was thus performed. Ionic products
arising from electron transfer
(ET), hydride transfer (HT), oxygenatom
transfer (OAT), and formal addition
(Add) may be observed, with a
predominance of two-electron processes,
whereas the product of hydrogenatom
transfer (HAT),
[(tpfpp)MnIVOH]+, is never detected.
A thermochemical threshold for the
formation of the product radical cation
allows an evaluation of the electrontransfer
ability of a MnV-oxo complex,
yielding a lower limit of 7.85 eV for the
ionization energy of gaseous
[(tpfpp)MnIVO]. Linear free-energy
analyses of the reactions of para-substituted
N,N-dimethylanilines and thioanisoles
indicate that a considerable
amount of positive charge is developed
on the heteroatom in the oxidation
transition state. Substrates endowed
with different heteroatoms, but similar
ionization energy display a comparable
reaction efficiency, consistent with a
mechanism initiated by ET. For the
first time, the kinetic acidity of putative
hydroxo intermediates playing a role in
catalytic oxidations, [(tpfpp)FeIVOH]+
and [(tpfpp)MnIVOH]+, has been investigated
with selected reference
bases, revealing a comparatively higher
basicity for the ferryl, [(tpfpp)FeIVO],
with respect to the manganyl,
[(tpfpp)MnIVO], unit. Finally, the neat
association reaction of 2 has been studied
with various ligands showing that
harder ligands are more strongly
bound.
"
* Autophagy;
* Degradative proteomics;
* Disease;
* Proteasome;
* Ubiquitin
Abstract
Protein degradation is a fundamental biological process, which is essential for the maintenance and regulation of normal cellular function. In humans and animals, proteins can be degraded by a number of mechanisms: the ubiquitin-proteasome system, autophagy and intracellular proteases. The advances in contemporary protein analysis means that proteomics is increasingly being used to explore these key pathways and as a means of monitoring protein degradation. The dysfunction of protein degradative pathways has been associated with the development of a number of important diseases including cancer, muscle wasting disorders and neurodegenerative diseases. This review will focus on the role of proteomics to study cellular degradative processes and how these strategies are being applied to understand the molecular basis of diseases arising from disturbances in protein degradation.