Proteomics/Metabolomics

BioSCENTer

Doctoral School
Technology Watch Course

E. Waelkens
Genomics oriented research

Transcriptomics
(micro-arrays)
Clinical oriented research

Biology oriented research

Proteomics
Physiology/Pharmacology oriented
research

Food oriented research

Bioinformatics
Chemistry oriented research Metabolomics
oriented research
Proteomics: What?

Proteins

Proteomics = the
study of the protein
library
- under certain well-defined
conditions
- in certain cells/tissues/
organisms
 Proteomics
 Genomics

Genomics =/=> Proteomics

Genes Proteins
 Proteomics

Proteins AA level

Protein level

•Proteolytic maturation
•Methionine processing
MODIFICATIONS !
•Sugars, Ubiquitination
•…….
 Metabolomics

Genes
Proteins

Metabolites
 Challenges in Proteomics/Metabolomics

• Dynamic changing (not static)

• Labile modifications (eg phosphorylation)

• Handling dependent (sample (pre)treatment !)

• Wide concentration range (no PCR equivalent)

• Localization dependent

• Better technology = more data

• Differential analysis and quantification

 Proteomics/Metabolomics:
MS Core technology
Proteomics ( 2 “soft ionization” techniques:
ESI and MALDI)
Metabolomics: FTMS

Mass spectrometry
 Proteomics: How?

Various ways

examples:

• (2D)- gel electrophoresis (old, old fashioned, but it still

climbs up the mountains)
• immunological techniques [western blot ,
immunohistochemistry, fluorescence,...]
• chromatography: UV profiling
• fluorescent tagged proteins [full- length,
(sub)cellular localization]
• Protein (Peptide) arrays
• Mass spectrometry
 Metabolomics: How?

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

MORE SEPARATION = MORE

IDENTIFICATION

 Combine separation steps: immuno-affinity based, subcellular

fractionation, serum depletion kits, ….
 Typical Proteomics
Separation techniques
- GEL based separations
• 1D Gel (SDS-PAGE)
• 2D GEL  2D DIGE
- Liquid Chromatography (LC)

- new separation techniques

 Separation

GEL based separations

• 1D Gel (SDS-PAGE)
• 2D GEL -> 2D DIGE

35

Biringer & Amato, 2002

 Separation

GEL based separations

• 2D DIGE = labeling with fluorescing cyanine dyes:

Cy2 (green) Cy3 (orange) Cy5 (red)

2D-DIGE,
 Separation

Liquid Chromatography (LC)

• 2D –Gel
 2D LC
• massive amount of data when linked to MS
 New Separation techniques

• Special affinity based separations (special affinity matrixes: eg

for serum samples/ membrane proteins/ phoshorylated peptides,
antibodies based separations, dendrimer capture). Popular
application: magnetic beads

• solid phase extraction

• new separations in the MS analyser itself: ion mobility

• new LC based separations:

• Advion Robot
• UPLC chip based separations
• robotic MALDI MS spotters
• mixed functional phase columns
 Separation

MORE SEPARATION = MORE

IDENTIFICATION

Note: MS identifications = routinely based on “bottom-up” strategy

(= peptides derived from the protein)

Holy grail: isolate 1 specific peptide from 1 protein

 Special Separation

- label proteins at rarely occurring AA’s (eg cysteine or methionine)

- analyze labeled peptides only --> reducing complexity

Analysis
Protein tryptic peptides

ICAT (isotope coded affinity tag) , COFRADIC

 Flow Chart

Separation

Analysis

Data processing
Analysis: History

Early Mass Spectrometer

 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 analyzer detector

Create ions Separate ions Detect ions

 Evolution MS technology

Huge improvement of mass spectrometers:

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

95 Multiple charged ions

90 +30
+34 968.4
85 854.6

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

Ions are preferentially +1

Special MALDI: SELDI
 Block 2. Analyzer

Important observation:
2 forces determine the behaviour
of the created ions:

• the mass (bigger = slower)

• the charge (higher = faster)

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

Linear (2D-) Model

2. Separate ions: Quadrupole MS
 2. Separate ions: Time of Flight

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

• mass accuracy (1–2 ppm)

• high resolving power (up to 200,000)
• High dynamic range
Separate ions: Ion Mobility device
Block 3. Detector

ION DETECTORS:
Channeltron
Daly detector (photomultiplier
conversion dynode)
Electron multiplier tube (EMT)
Faraday cup
Microchannel plate
 Block 3. Detector

Example: dynode detector

 Basic Proteomics MS instrument

source analyzer detector

ESI or MALDI e.g. IonTrap

or TOF (time of flight)

Examples: •ESI IonTrap

•MALDI TOF
 More complex MS instruments

source analyzer analyzer detector

ESI or MALDI e.g. quadrupole e.g. quadrupole
or time-of-flight or TOF or Iontrap

Collision cell

• ESI Q TOF
Examples:
• ESI Q Iontrap
• MALDI Iontrap
• MALDI TOF TOF
 MS/MS: AA sequence

Note: manual interaction or de novo sequence

MS/MS of m/z 730.4

ALGSFR
 FT-MS: Metabolomics

source analyzer detector

ESI or MALDI FT based

 Empirical formula (composition)
Azo_1 #257-284 RT: 4.99-5.27 AV: 17 NL: 1.54E6
T: FTMS - p NSI Full ms [ 85.00-1000.00]
241.1199
C 11 H 17 O 4 N 2
C 12 H 13 N 6
100

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

From real life…..

MALDI-TOF/TOF
Triple Q - IonTrap
Q - TOF
 MALDI-IonTrap
Laser
Ion Transfer
Tube

Skimmer
+
+
+
+
+
Tube Lens
FT-MS
 Top Down Analysis

Sequence Analysis of m/z 893

928.6396

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+

726.1155 1161.3905 1451.3701

1064.8219
682.5838 1175.4320 1301.2075 1463.2484
 Top Down Analysis

928.6396

MS/MS of m/z 893

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

600 700 800 900 1000 1100 1200 1300 1400

m/z

11124.3263
100

90 Zero Charge Monoisotopic Data

80

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

How to find differences ?

2 different strategies:

Labeling

Label free
 Example 1: Biomarkers

Labeling Based on stable isotopes (not radioactive): C12  C13

N14  N15

Examples: SILAC (stable isotope labeling )

ICAT
unbiased
ITRAQ
O18 labeling
AQUA (=targetted approach)
 Labeling: SILAC

Stable isotope labeling with amino acids in cell culture (SILAC)

eg: 13C labeled L-lysine :

- 2 populations (“light” and “heavy” amino acid in cell culture)

12C L-lysine 13C labeled L-lysine

Duplex Labeling technique

Labeling in vivo
 Labeling: ICAT

2 labels:

LINKER C12 BIOTIN

LINKER C13 BIOTIN

 Labeling: ICAT

reacts with cysteines

2 labels:

LINKER C12 BIOTIN

LINKER C13 BIOTIN

 ICAT

LINKER C12 BIOTIN

LINKER C13 BIOTIN

Step 1: label proteins

Step 2: digest proteins

 ICAT

LINKER C12 BIOTIN

LINKER C13 BIOTIN

Step 2: digest proteins

 ICAT

LINKER C12 BIOTIN

LINKER C13 BIOTIN

Step 3: isolate labeled peptides

 ICAT

Step 4: remove Biotin tag (= cleavable

Isotope-Coded Affinity Tag (cICAT))

LINKER C12 BIOTIN

LINKER C13 BIOTIN

 Summary ICAT labeling

ICAT (Isotope-coded affinity tag)

= Duplex Labeling:
2 tags (“light” and “heavy” linker, 9x C12 and 9x C13, ∆ mass= 9 Da)

has a biotin tag

labeling = at the protein level (at cysteines)

analysis = only labeled peptides  reduced complexity

LINKER C12 BIOTIN

LINKER C13 BIOTIN

 Labeling: iTRAQ
Isobaric Tag
(Total mass = 145)

Reporter Balance PRG

Charged Neutral loss

 Gives strong signature ion in MS/MS

 Gives good b- and y-ion series
 Maintains charge state
 Maintains ionization efficiency
 Signature ion masses lie in quiet region

Reporter Balance Peptide Reactive

(Mass = 114 thru 117) (Mass = 31 thru 28) Group

= MS/MS Fragmentation Site

 iTRAQ: Multiplexing

114 31 PEPTIDE

115 30 PEPTIDE
MIX  MS

116 29 PEPTIDE 1352.84

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

1347.0 1349.6 1352.2 1354.8 1357.4 1360.0

Mass (m/z)

1 M/Z peak !
100
5186.0

90

80

70

60

50

MS/MS % Intensity

40

30

20

10

111.0 112.8 114.6 116.4 118.2 120.0

Mass (m/z)

4 # fragments !
iTRAQ

Reporter Group Placement:

Selection of ‘Quiet Region’
 iTRAQ: Absolute Quantification

Xrn1 ∆ Upf1 ∆ wt
lysate lysate lysate

Reduce/ Reduce/ Reduce/

Block Cysteines Block Cysteines Block Cysteines

Synthetic
Trypsin digest Trypsin digest Trypsin digest
peptides

React with React with React with React with

iTRAQ™ Reagent iTRAQ™ Reagent iTRAQ™ Reagent iTRAQ™ Reagent
114 115 116 117

MIX

• Single 2D LC analysis • Add at known

SCX
for combined samples concentration
(4-plex)
LC - MS/MS
 Labeling: AQUA

Digestion

AQUA peptide: known

concentration, isotope labeled

Set parent ion Set fragment ion

MIX

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

DATA Processing !!!!

 Example 2: Biomarkers

Label-free

2 examples:
- Matching the data using gel-imaging software
- relative quantification of candidate biomarkers using MRM
 2D Gel view

Label-free “GEL” view matching approach

2D GEL view: mapping using retention time and m/z value

 Label-free: MRM approach

Digestion
MRM= Multiple Reaction Monitoring

Set parent ion Set fragment ion

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

• high dynamic range of protein concentrations

• dominated by a limited number of high-abundance proteins, constituting
up 95–99% of the total protein

Depletion methods (2-20 proteins):

Immunodepletion methods
Affinity methods
- old methods: Cibacron Blue (Blue Sepharose): albumin; Protein G
(A): IgG
- new methods: combinatorial chemistry: eg short peptide fragments
(hexapeptides)
Example 3: Serum Proteomics
 Example 3: Serum Proteomics

Low Abundant

High Abundant

Up to 6 fold increase in identification !

 Example 4: Tissue proteomics
using MALDI MS

Matrix Coating on Tissue

Optical Image
MALDI Imaging Mass Spectrometry
 Tissue Proteomics using IMS

Tissue Biomarker Discovery Alterations in Hippocampus:

KO mouse

“Fingerprint of striatum” Alterations in Hippocampus:

WT mouse
 To remember

Proteomics/Peptidomics/Metabolomics

- Many possible approaches

- More separation = more identification
- For serum/plasma proteomics: use depletion methods
- Biomarker discovery: start with the affected tissue
- Advanced dataprocessing = indispensable
 Future perspectives

genomics + proteomics + peptidomics + metabolomics + …

 Personalized medicine
Proteomics Metabolomics

Bioinformatics

Total spectrum analysis

Sample Separation:
Liquid Chromatography
GE Healthcare Ettan MDLC (left)
Dionex/LC Packings Ultimate 3000 (right)
 Guided tour: Equipment

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

Support protein and peptide separations, interpretation of results

Protein digestions
MALDI MS protein/peptide mass measurements (linear/reflectron/+-)
ESI MS protein/peptide mass measurements
FTMS-ESI mass measurements
MALDI MS/MS protein/peptide identifications
ESI MS/MS protein/peptide identifications
1D LC ESI MS
2D LC ESI MS
1D LC MALDI MS
2D LC MALDI MS

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.

Services (some examples) Research (some examples)

Mass Analysis Biomarker Discovery

Solitary Form of S. Gregaria

Same species
Gregarious Form of S. Gregaria

Protein Identification Imaging Mass Spectrometry

 Contact

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