Sept30 EW 2
Sept30 EW 2
Sept30 EW 2
BioSCENTer
Doctoral School
Technology Watch Course
E. Waelkens
Genomics oriented research
Transcriptomics
(micro-arrays)
Clinical oriented research
Proteomics
Physiology/Pharmacology oriented
research
Proteins
Proteomics = the
study of the protein
library
- under certain well-defined
conditions
- in certain cells/tissues/
organisms
Proteomics Genomics
Genes Proteins
Proteomics
Proteins AA level
Protein level
•Proteolytic maturation
•Methionine processing
MODIFICATIONS !
•Sugars, Ubiquitination
•…….
Metabolomics
Genes
Proteins
Metabolites
Challenges in Proteomics/Metabolomics
• Localization dependent
Mass spectrometry
Proteomics: How?
Various ways
examples:
Various ways
examples:
• biochemical assays
• GC- MS
• LC- MS
• CE- MS
• LC- NMR
• LC- EC (electrochemical array)
• FT-ICR- Mass spectrometry
Flow Chart
Separation
Analysis
Data processing
Separation
• 1D Gel (SDS-PAGE)
• 2D GEL -> 2D DIGE
35
2D-DIGE,
Separation
• 2D –Gel 2D LC
• massive amount of data when linked to MS
New Separation techniques
• Advion Robot
• UPLC chip based separations
• robotic MALDI MS spotters
• mixed functional phase columns
Separation
Analysis
Protein tryptic peptides
Separation
Analysis
Data processing
Analysis: History
obtained from:
Yergey A.L., Yergey A.K.
Preparative Scale Mass Spectrometry: A Brief History of the Calutron
JASMS, 1997, V8(N9), p943-953
Some Milestones
1897: Sir J.J Thomson, Cavendish Laboratory , University of Cambridge, discovery of the electron
1906: Thomson: Nobel Prize for this studies on the conduction of electricity by gasses (ion movements)
1912: Thompson: First mass spectrometer
1946: William E. Stephens: concept of time-of-flight analyzer
1953-58: Wolfgang Paul: quadrupole analyzer
1983: first commercial ion trap
1989: Paul: Nobel Prize in Physics
1968: Malcolm Dole: first concept of ESI
1974: Comisarow & Marshall: FTMS
1984-88: John Fenn: ESI for biomolecule analysis
Wong, S.F., Meng, C.K. and Fenn, J.B., J. Phys. Chem., 92, 546 (1988)
Meng, C.K., Mann, M. and Fenn, J.B., Z. Phys.D., 10, 361 (1988)
1988: Tanaka (Japan) and Franz Hillenkamp/Michael Karas (Germany): MALDI
M. Karas and F. Hillenkamp, Anal. Chem. 60, 2299-2301 (1988)
K. Tanaka et al., Rapid Commun. Mass Spectrom. 2, 151-153 (1988)
Basic MS instrument
source
- end 80’s: Soft ionization methods : ESI and MALDI (Nobel price 2002)
- end 90’s: hybrid MS instruments, nanospray, MALDI TOF/TOF
analyzer
- begin 21 century: exponential growth of MS equipment:
TOF/TOF with real CID, linear iontrap, orbitrap, 9.4 Tesla FT ICR MS, ECD
and ETD dissociation, new hybrids (eg MALDI-Iontrap; 3Q-Iontrap..),
ion mobility
data processing
- CPU power
Block 1: Ionization
IONIZATION METHODS:
Chemical ionization (CI)
Electron impact (EI)
Electrospray ionization (ESI)
Fast-atom bombardment (FAB)
Field ionization
Laser ionization (LIMS)
Matrix-assisted laser desorption ionization (MALDI)
Plasma and glow discharge
Plasma-desorption ionization (PD)
Resonance ionization (RIMS)
Secondary ionization (SIMS)
Spark source
Thermal ionization (TIMS)
1: Electrospray Ionization (ESI)
Special ESI: Nanospray
Wilm, 1991
Special ESI: Nanospray
Electrospray Ionization (ESI)
+31
Averaged spectrum of 2 components
+33 +32 937.2
100 880.5 907.9
80
+29
75 +35 1001.7
830.3
Relative Abundance
70 +36
807.3 +28
65 1037.5
60
55 +27
1075.8
50 +26
+37 1117.1
45 785.5
+25
40 1161.7
35 +38
764.8
30
+39
25 745.3
20
+ 40
15 726.6
10
5
0
750 800 850 900 950 1000 1050 1100 1150 1200
m /z
Deconvolution
Deconvoluted Spectrum
Carbonic Anhydrase
29021.0
100 Reported MW1 = 29,022
95
90 Yeast Enolase
85 Reported MW1 = 46,671
80
75
Relative Abundance
70
65 46668.0
60
55
50
45
40
29105.0
35
30 47085.0
25
20
15
10
5
0
5000 10000 15000 20000 25000 30000 35000 40000 45000
mass
MALDI ionization
MALDI ionization
MALDI
MALDI ionization
MALDI
Important observation:
2 forces determine the behaviour
of the created ions:
Therefore:
molecule with mass 1000 Da but charge +2, runs as a molecule
with mass 500 Da but with charge +1 m/z (not just ‘m’)
Block 2. Analyzer
• Ion-trap MS
• Quadrupole MS
• Time-of-flight MS
• Fourier-transform MS
• Magnetic-sector MS
• Orbitrap MS
• Ion mobility MS
2. Separate ions: Ion-Trap
esilcq.exe
3D-Model
12
Laser
9 3
6
2. Separate ions: Time of Flight
12
9 3
6
2. Separate ions: Fourier-Transform
LTQ_FT.exe
2. Separate ions: Magnetic-Sector
Separate ions: Orbitrap analyzer
ION DETECTORS:
Channeltron
Daly detector (photomultiplier
conversion dynode)
Electron multiplier tube (EMT)
Faraday cup
Microchannel plate
Block 3. Detector
Collision cell
• ESI Q TOF
Examples:
• ESI Q Iontrap
• MALDI Iontrap
• MALDI TOF TOF
MS/MS: AA sequence
ALGSFR
FT-MS: Metabolomics
90
181.0509
C9 H 9 O4 227.1042
80
C 10 H 15 O 4 N 2
C 11 H 11 N 6
70
Relative Abundance
60
50
40
135.0454
C8 H 7 O2 195.0667
30 C 10 H 11 O 4 216.0519
174.0564 C 8 H 10 O 6 N 1
C 10 H 8 O 2 N 1 259.0766
20 C7 H 4 O1 N8 C 18 H 11 O 2
C 4 H 13 O 8 N 5
10 168.0431
C8 H8 O4
103.3712 132.0555 145.9380
0
120 140 160 180 200 220 240 260
m/z
Some Examples
Skimmer
+
+
+
+
+
Tube Lens
FT-MS
Top Down Analysis
893.5290
Relative Abundance
929.9735 MS/MS of m/z 893
720.3784
823.9249
13+ 769.9116
916.4733
955.7405
1008.5148
1097.6791
676.8584
900.2117 1229.5820 1358.6410
588.3018 663.0114 1115.5698
1239.3015 1348.3876 1412.5938
600 700 800 900 1000 1100 1200 1300 1400
m/z
893.6815
14+
12+
829.7762
967.9076
829.4963
11+
1055.8090
15+
774.5244
10+
928.6396
929.9735
Relative Abundance
720.3784
823.9249
916.4733
769.9116
955.7405
1008.5148
1097.6791
676.8584
900.2117 1229.5820 1358.6410
588.3018 663.0114
1115.5698
1239.3015 1348.3876 1412.5938
11124.3263
100
70
60
Relative Abundance
50
3713.7782
40
30
8769.1763
4027.9395
5079.5063 9822.7211
20 1700.8490
7947.8305 10058.8464
10 2778.3347 4738.4032 9667.6773
6858.2459
0
1000 2000 3000 4000 5000 6000 7000 8000 9000 10000 11000
Flow Chart
Separation
Analysis
Data processing
Data Processing
• Protein/peptide/metabolite identification =
search engines to interrogate database(s)
• high throughput = gigabytes of data
• “meta”-processing of data: PTM analysis,
clustering, differential analysis, pathway
analysis …..
Advanced Data processing
Advanced Data Processing
Hierarchical Clustering
Female
Male
Advanced Data Processing
PCA Classification
male
female
Example 1: Biomarkers
• Diagnostic
• Prognostic
• Therapeutic
Example 1: Biomarkers
2 different strategies:
Labeling
Label free
Example 1: Biomarkers
2 labels:
= Duplex Labeling:
2 tags (“light” and “heavy” linker, 9x C12 and 9x C13, ∆ mass= 9 Da)
114 31 PEPTIDE
115 30 PEPTIDE
MIX MS
117 28 PEPTIDE
1347.0 1349.6 1352.2 1354.8 1357.4 1360.0
Mass (m/z)
1 M/Z peak !
145
iTRAQ: Multiplexing
1352.84
1 M/Z peak !
100
5186.0
90
80
70
60
50
MS/MS % Intensity
40
30
20
10
Mass (m/z)
4 # fragments !
iTRAQ
Xrn1 ∆ Upf1 ∆ wt
lysate lysate lysate
Synthetic
Trypsin digest Trypsin digest Trypsin digest
peptides
MIX
Digestion
MRM analysis
Source Q1 Q3
Q2
Example 1: Biomarkers
Labeling
• At protein level or at peptide level
• relative quantification and absolute quantification (using standards)
• allows MULTIPLEXING
Note:
2D DIGE = also multiplexing and relative quantification
Example 2: Biomarkers
Label-free NO MULTIPLEXING
Label-free
2 examples:
- Matching the data using gel-imaging software
- relative quantification of candidate biomarkers using MRM
2D Gel view
Digestion
MRM= Multiple Reaction Monitoring
Source Q1 Q3
Q2
Example 2: Biomarkers
Label-free
• At protein level or at peptide level
• only relative quantification (using standards)
• no MULTIPLEXING
• Cost effective and easy
Examples:
SELDI-MS
2D LC MS/MS with mapping of the data
using retention time and m/z value
MRM methods
Example 3: Serum Proteomics
Low Abundant
High Abundant
Optical Image
MALDI Imaging Mass Spectrometry
Tissue Proteomics using IMS
Proteomics/Peptidomics/Metabolomics
Personalized medicine
Proteomics Metabolomics
Bioinformatics
ESI MS
Thermo Electron LTQ
Analytical Instruments:
Mass Spectrometry (single and tandem)
ESI: Thermo Electron LTQ / Bruker MicroTof Q
MALDI: Bruker UltraFlex II
ESI+MALDI: Bruker FT-MS 9.4 Tesla
Guided tour: Equipment
MALDI TOF/TOF MS
Bruker UltraFlex II
Analytical Instruments:
Mass Spectrometry (single and tandem)
ESI: Thermo Electron LTQ / Bruker MicroTof Q
MALDI: Bruker UltraFlex II
ESI+MALDI: Bruker FT-MS 9.4 Tesla
Guided tour: Equipment
FT-MS
Bruker FT-MS 9.4 Tesla
Analytical Instruments:
Mass Spectrometry (single and tandem)
ESI: Thermo Electron LTQ / Bruker MicroTof Q
MALDI: Bruker UltraFlex II
ESI+MALDI: Bruker FT-MS 9.4 Tesla
Guided tour: Equipment
Additional instruments
Proteineer FC Robot (MALDI target plate spotter)
Some workflows
Proteome profiling
Metabolites, small organic compounds
Metabolomic profiling (LC-MS)
Data processing : PCA, Biomarker discovery……
Database searching
What are ProMeta’s tasks?
• Services:
Provide state-of-the-art proteomics and metabolomics analysis services to
groups inside and outside the K.U.L. and to third parties.
• Research:
Advance proteomics and metabolomics research via internal research projects.
Same species
Gregarious Form of S. Gregaria
Coordinator:
• Daily governing of ProMeta-facility: Dr. Geert Baggerman
• 016/ 330458 (Office) 330454 (Lab)
•Website: www.prometa.kuleuven.be
Acknowledgements
Prof R. Derua
Prof J. Goris
Prof V. Janssens
Prof B. De Moor
Prof J. Van Lint
Prof J. Vandenheede R. Van de Plas
E. Ivanova
C. Lambrecht
J. Louis
W. Sents
K. Schildermans
R. Verbiest
S. Vandoninck
Thank You !
www.Prometa.be
BioMacS.kuleuven.be
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