PAF introduction

The PAF (also called UNIL Proteomics Platform) is a research and service core facility devoted to the high-performance analysis of proteins

•Service :

•Identification of gel-separated proteins by MS

•Project discussion

•2D-PAGE and/or other multi-dimensional separation techniques

Research :

•Development of proteomics methods

•Applications to biological problems

•Teaching :

•Techniques, possibilities & limitations of proteomics approaches

PAF technology and instrumentation

Nano-HPLC- Quadrupole-time-of-flightMass spectrometer

Nano-HPLC-triple quadrupole ion trapMass spectrometer

2D-electrophoresis1D-electrophoresis

260404RPChistonesFRAC001:1_UV1_280nm 260404RPChistonesFRAC001:1_Conc 260404RPChistonesFRAC001:1_Fractions 260404RPChistonesFRAC001:1_Inject

-30.0

-20.0

-10.0

0.0

10.0

20.0

30.0

mAU

0.0 5.0 10.0 15.0 20.0 25.0 ml

A1 A2 A3 A4 A5 A6 A7 A8 A9A10 A11 A12 B12 B11 B10 B9 B8 B7 B6 B5 B4 B3 B2 B1 C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 C12 D12 D11 D10 D9

Liquid Chromatography

New : access to ABI 4700 TOF/TOF™ (courtesy Dept. Biochemistry)

MALDI - Tandem Time of Flight Mass Spectrometer for High Throughput Protein identification

Challenges of in vivo proteomics

• Complexity : – 35’000 genes (?) in H. sapiens, – 15’000 expressed in a single cell ? – but >50’000 chemically different protein species ?

• Dynamic range :– 105 x or 106 x between low and high-abundance proteins

• Plasticity : – continuous variation in protein expression pattern (every s),

PTM’s, degradation,…

PTM analysisPTM analysis

Interaction / Functional Interaction / Functional ProteomicsProteomics

Protein expression Protein expression analysisanalysis

FOCUS : Subcellular fractionOrganelleProtein Complex20-200 proteins

FOCUS : Complex samplesWhole proteomes200 and more proteins

FOCUS :Single protein

1-20 proteins

Sample complexity

Analytical Detail

Overview : classes of proteomics experiments

Proteomics

• 2D-PAGE

• Mass Spectrometry for proteomics

• Proteomics workflows and applications

2D-PAGE

How to create a pH gradient ?

IEF: the principle

Improvements in 2D-PAGEImprovements in 2D-PAGE

-+

- IPG (Immobilised Ph Gradient) strips for the first dimensionpH-forming chemical groups are grafted onto the

polyacrylamide matrix, creating a mechanically stable pH gradient

+ + mechanical stability+ + reproducibility+ + loadable amounts+ + „zoom“ pI ranges

pH 3.0 10.0

Equilibration step 1 : •SDS•Buffer pH 6.8•DTT (reduce –S-S-)

Equilibration step 2 : •SDS•Buffer pH 6.8•Iodoacetamide (alkylate –S-S-)

After IEF : equilibration and 2nd dimension

+ -

6.5 10 3.0 5.5 8.5

pI

100

75

50

37

2010

150

Database of 2D images with clickable spots :

www.expasy.org/ch2d/

One protein many spots

Post-translational modifications can result in changes in pI and/or MW

Spot “trains” for extensively modified proteinsCaused by

GlycosylationPhosphorylationAcetylation (K)…

2D-PAGE and image analysis are used for studying changes in composition of the

proteome

Control

Stimulus applied

•Spot detection

•Spot quantification

•Gel-to-gel•Matching

•Presence/absenceof spot

•Up/down regulation

•Statistic analysis

Software-based image analysis

Mass Spectrometry in proteomics

Mass spectrometry : essential functions

SAMPLEION

GENERATIONION

SEPARATIONION

DETECTION

ION SOURCE MASS ANALYZER DETECTOR

ESI : Electrospray Ionisation

MALDI : Matrix Assisted Laser Disorption/Ionization

QuadrupolesIon trapsTime-of-flight with reflectronTOF/TOFFT-ICR (Fourier transform –Ion Cyclotron Resonance)

Faraday cupScintillation counterElectromultiplierHigh-energy dynodes with electronmultiplierArray (detector)FT-MS

Masses and mass measurements

•All mass spectrometers function measure molecules in their ionized state

•All values determined by MS are relative to the m/z assumed by the molecule after the ionization process

The relationship between the molecular mass (m) and the m/z value can be calculated as follows:

m/z = (m + (mA * z )) / z

mA is the mass of the adduct responsible for ionization (typically H+ for positive MS mode).

MALDI (Matrix Assisted Laser Desoprtion Ionisation

MALDI TOF (Time Of Flight)

MALDI IONISATIONMALDI IONISATION

• Great for Peptide Mass Fingerprinting– Fast– Easy to measure – Sensitive– Salt-tolerant (to some extent)– Also good for larger MW (small proteins)– Sample on a stable support (no time constraints)– 1+ ions simpler data analysis

•High accuracy needs careful calibration•Difficult (but possible) to do MS/MS by MALDI•Signal suppression in complex mixtures•Crystallisation conditions influence results

disadvantages

MALDI TOFMALDI TOF

MALDI-TOF of a tryptic digest of BSA

+TOF MS: 50 MCA scans from Sample 1 (BSA Digest 100 fmol) of BSA Digest 100 fmol MS ...a=3.56217430068478150e-004, t0=3.64725878201043440e+001, Thresholded

Max. 1305.0 counts.

800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800m/z, amu

0

10

20

30

40

50

60

70

80

90

100

110

120

130

140

150

160

170

180

190

Inte

ns

ity, c

ou

nts

927.59

847.59

1440.00 1479.98 1567.94

1640.161022.56869.07

1305.871163.77 1481.98

1296.861249.771050.55871.07 1024.56 1417.931283.91789.53 1142.86 1443.011073.03 1595.951386.76 1824.09857.14

1108.71 1292.95978.60 1790.101501.84 1616.92

YLYEIAR

LSQKFPK LVNELTEFAK

FKDLGEEHFK

HPEYAVSVLLR

HLVDEPQNLIK

LGEYGFQNALIVR

KVPQVSTPTLVEVSR

DAFLGSFLYEYSR

??

?

? ??

P. Kebarle, M. Peschke / Analytica Chimica Acta 406 (2000) 11–35

• molecules compete for ionisatione.g. Na+ >> Peptide

ELECTROSPRAY IONISATIONELECTROSPRAY IONISATION

• Great for MS/MS– Can be directly coupled to reversed phase LC

(separation !)– Sensitive– Excellent for MS/MS due to 2+/3+ ions

•Sample introduction more complex•Data analysis more difficult (2+/3+ ions) •one-shot sample analysis (time constraints)•Very low tolerance to contaminants

disadvantages

ELECTROSPRAY IONISATIONELECTROSPRAY IONISATION

1+ versus 2+ ions

1160 1162 1164 1166 1168 1170 1172 1174m/z, amu

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

8084

Intensity, counts

1163.76

1164.76

1165.74

1166.781162.70

m=1.0 Da

1.0 Da

1.0 Da

1+

580.0 581.0 582.0 583.0 584.0 585.0 586.0m/z, amu

0

20

40

60

80

100

120

140

160

180

200

Intensity, counts

582.28

582.77

583.27

m=0.5 Da

0.5 Da

0.5 Da

2+

Modes of measurement : MS & MS/MS

•Ion production (ionisation)

•Ion separation

•Ion detection

•Ion production (ionisation)

•Ion separation – isolation of “parent” ion

•Ion fragmentation (CID)

•Ion separation – separate fragment ions

•Ion detection - measure fragment ions

MS

Tandem MSMS/MS

Tandem MS (MS/MS) facts

• MS/MS results in the acquisition of pseudo-sequence information for multiple (as many as possible) tryptic peptides in addition to intact peptide mass information

• MS/MS needs an instrument able to perform ion isolation and fragmentation, in addition to regular MS

• MS/MS results in the acquisition of multiple orthogonal data files (1 spectrum / peptide)

•CID= collision induced dissociation

•Low energy (<100 eV) vs high energy collisions (>> 100 eV)

•Precursor ion = parent ion : the one being fragmented

•Daughter ions = fragment ions produced by CID

•Tandem mass spectrometry = MS/MS•- here : the combination of ion selection / CID / fragment analysis

•ESI of tryptic peptides typically generates doubly charged ions due to the presence of Lys or Arg at the C terminal end of the peptides

•y and b-ion series fragments are usually observed in MS/MS fragmentation spectra.

•CID= collision induced dissociation

•Low energy (<100 eV) vs high energy collisions (>> 100 eV)

•Precursor ion = parent ion : the one being fragmented

•Daughter ions = fragment ions produced by CID

•Tandem mass spectrometry = MS/MS•- here : the combination of ion selection / CID / fragment analysis

•ESI of tryptic peptides typically generates doubly charged ions due to the presence of Lys or Arg at the C terminal end of the peptides

•y and b-ion series fragments are usually observed in MS/MS fragmentation spectra.

MS/MS Glossary and facts

Covalent bonds being broken ion series

Covalent bonds being broken ion series

ESI & Quadrupole-based instruments

Triple Quadrupole

Triple Quadrupole-Ion trap

Quadrupole-QuadrupoleTOF

Ion Trap (3D trap)

All these instruments can perform MS/MS fragmentation experiments

• Identifying proteins by mass spectrometry

MS-based protein identification : general concept

Protein sample

Protein fragments (5-30 AA peptides)

Exact masses of peptides

Fragmentation (MS/MS)spectrum of each peptide

Protein sequence(s)

Protein fragment sequences (same protease specificity)

Calculated exact masses of peptides

Calculated fragmentation spectrum of each peptide

Specific proteasee.g. trypsin

MS

software

software

software

Best Match(es)

experimental In silico

• Protein identification by Peptide Mass Fingerprinting (PMF)

+TOF MS: 50 MCA scans from Sample 1 (BSA Digest 100 fmol) of BSA Digest 100 fmol MS ...a=3.56217430068478150e-004, t0=3.64725878201043440e+001, Thresholded

Max. 1305.0 counts.

800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800m/z, amu

0

10

20

30

40

50

60

70

80

90

100

110

120

130

140

150

160

170

180

190

Inte

nsity

, co

un

ts

927.59

847.59

1440.00 1479.98 1567.94

1640.161022.56869.07

1305.871163.77 1481.98

1296.861249.771050.55871.07 1024.56 1417.931283.91789.53 1142.86 1443.011073.03 1595.951386.76 1824.09857.14

1108.71 1292.95978.60 1790.101501.84 1616.92

Extracted peak list

m/z

847.5896869.0722922.5712923.5815927.59041022.55511050.55331163.76951164.75311193.73931249.77051250.81031296.85561297.84991305.86681416.89291440.00081479.97731482.95831567.94171640.16351824.06…

Information contained in MS spectrum

Search form…

Results page…

• Protein identification by MS/MS

Experimental set-up : nanoLC-MS/MS

database correlation

C18 ColumnL = 10 cmID = 50-100 µm

Mass spectrometer

T-splitter

~5%

HPLCpumps

~95%further analysis

(waste)sample

16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31

Time, min

0.0

4000.0

8000.0

1.2e4

1.6e4

2.0e4

2.4e4

2.8e4

3.2e4

3.6e4

Intensity, cps

568.58

496.57 563.23354.11445.07

+TOF Product (461.7)

50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900m/z, amu

0

5

10

15

20

Intensity, counts

86.09

147.01

635.31260.17175.06

270.11522.23

748.39359.01

y8y7

y6

y5

y4

y3

y2y1

SSILFDIK

461.71

375.19

An on-line LC-ESI-MS experiment with automatic data acquisitionAn on-line LC-ESI-MS experiment with automatic data acquisition

Mr peptide : 921.4

MeCNgradient

25 30 35 40 45 50 55 60 65 70 75 80Time, min0.00

5.00e4

1.00e5421.68

653.32464.17 582.25

722.28 501.73507.75

740.41595.29 700.34

Chromatogram : Total ion current vs. time

MS/MS peptide 4 : +TOF Product (662.8):

10.0

14.0

100 200 300 400 500 600 700 800 900 1000 1100 1200 1300

m/z, amu

0.0

5.0

235.08

166.06147.10

262.10

120.06409.18207.09

663.3252110.04

614.2136

138.06

Full scan : +TOF MS:

m/z, amu

400 450 500 550 600 650 700 750 800 850 9000

200

400518.82

777.8176536.80

662.76 531.47

507.1541

1553 (2+)

1553 (3+)1

2

34

Automated LC- MS/MS run

+TOF Product (653.3)

100 200 300 400 500 600 700 800 900 1000 1100 1200 1300m/z, amu

0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

9.0

10.0

11.0

12.0

13.0

14.0

15.0

16.0

17.0

18.0

19.019.7

Intensity, counts

251.12

110.06

223.13

138.05 653.36

332.21

594.2586.09

350.24 712.461055.49

373.30 566.32465.16 956.41 1168.56166.05 841.53

206.16

a1

b1

I

a2

b2

y10

Ib3 y9

b4 y8a5

b5

y7

y6

b8

y3

Iy2

y1

Residue Immonium a b y-----------------------------------------------------------------------------------H, His 110.07 110.07 138.06 1305.71L, Leu 86.09 223.15 251.15 1168.65V, Val 72.08 322.22 350.21 1055.57D, Asp 88.03 437.25 465.24 956.50E, Glu 102.05 566.29 594.28 841.47P, Pro 70.06 663.34 691.34 712.43Q, Gln 101.07 791.40 819.39 615.38N, Asn 87.05 905.44 933.44 487.32L, Leu 86.09 1018.53 1046.52 373.28I, Ile 86.09 1131.6157 1159.61 260.19K, Lys 101.10 1259.7106 1287.70 147.11

MH22+

precursor

Matching of MS / MS data

Black : predictedRed : predicted and detected

Mascot search outputMascot search output

Mascot Search Results

Significant hits: ALBU_BOVIN (P02769) Serum albumin precursor (Allergen Bos d 6). ALBU_CANFA (P49822) Serum albumin precursor (Allergen Can f 3). VWF_PIG (Q28833) Von Willebrand factor precursor (vWF) (Fragment). CIQ3_BOVIN (P58126) Voltage-gated potassium channel protein KQT-like 3 RYR2_RABIT (P30957) Ryanodine receptor 2 (Cardiac muscle-type ryanodin K2CA_BOVIN (P04263) Keratin, type II cytoskeletal 68 kDa, component IA ALFB_RABIT (P79226) Fructose-bisphosphate aldolase B (EC 4.1.2.13) (Li

ALBU_BOVIN Mass: 71244 Total score: 711 Peptides matched: 12 (P02769) Serum albumin precursor (Allergen Bos d 6).    Observed   Mr(expt)   Mr(calc)   Delta  Miss Score  Rank   Peptide  

15     461.80     921.58     921.48      0.10    0     55     1    AEFVEVTK  19     501.80    1001.58    1001.58     0.01    0     31     1    LVVSTQTALA  31     569.80    1137.58    1137.49     0.09    0     72     1    CCTESLVNR  33     582.30    1162.58    1162.62    -0.04    0     76     1    LVNELTEFAK  47     653.40    1304.78    1304.71     0.08    0     90     1    HLVDEPQNLIK  58     722.40    1442.78    1442.63     0.15    0     87     1    YICDNQDTISSK  60     739.80    1477.58    1477.52     0.07    0     43     1    ETYGDMADCCEK  61     740.40    1478.78    1478.79    -0.00    0     61     1    LGEYGFQNALIVR 64     751.90    1501.78    1501.61     0.18    0    37     1    EYEATLEECCAK  74     547.30    1638.88    1638.93    -0.05    1     85     1    KVPQVSTPTLVEVSR  97     627.70    1880.08    1879.91     0.16    0     34     1    RPCFSALTPDETYVPK  98     636.70    1907.08    1906.91     0.16    0     42     1    LFTFHADICTLPDTEK

Orthogonal datasets and confidence levels

Database : 100’000 sequences 500 spectra

Probability of one (any) spectrum “accidentally” matching a sequence (wrong match) : 1/100’000 x 500 = 5.10-3 (0.005)

Probability of 2 spectra “accidentally” matching the same sequence (wrong match) : 5.10-3 x 5.10-3 = 2.5 x 10-5

Much higher confidence of identification with at least two peptides matching the same protein sequence

Every peptide is unambiguously assigned to its “parent “ sequence, therefore many proteins can be identified in one sample during one run

PMF :

one MS spectrum one dataset (peak list)

MS/MS :

n MS/MS spectra n orthogonal datasets

m/z

time

Chromatographic Separation (reversed-phase)

Tandem mass spectra of 50-2000 peptides

Q7Z5Y2 Mass: 118789 Total score: 178 Peptides matched: 6 Rho-interacting protein 3.  Mr(calc) Score Peptide  930.48 42 EGLTVQER  1032.54 11 NWIQTIMK 1206.63 29 FSLCILTPEK1369.75 24 LSTHELTSLLEK 1406.77 55 FFILYEHGLLR 1775.88 16 QVPIAPVHLSSEDGGDR

ProteasedigestionPeptideextraction

Nano-HPLC

MS/MS

Summary : Typical Analytical Workflow

Database matchesDHX9_HUMAN ATP-dependent RNA helicase ANFM_HUMAN Neurofilament triplet M proteinQ9BQG0 Hypothetical proteinMYO6_HUMAN Myosin VI. TP2A_PIG DNA topoisomerase II, alpha isozymeQ7Z5Y2 Rho-interacting protein 3. FLIH_HUMAN Flightless-I protein homolog. TP2B_MOUSE DNA topoisomerase II, beta isozymeS3B1_HUMAN Splicing factor 3B subunit Q8VCW5 Similar to alpha internexin neuronal Q8CHF9 MKIAA0376 protein (Fragment).

Protein sequence database

Database searching Software (MASCOT)Output :

•Protein identification in simple/complex mixtures

•Extensive sequence coverage and peptide mapping

•Analysis of modified peptides possible

Biological question

Caveat : Protein identification IS NOT

protein characterisation

Two peptides are enough to identify a proteinBut

We are still identifying two peptides, not the entire protein

Highly similar sequences cannot bedistinguished

For finding PTMs extensive Sequence coverage is essential !!

2 Technology, workflows and

applications : what is available

New and old tools

Protein separation techniques- Liquid chromatography- Electrophoresis -…

Protein identification techniques- Mass Spectrometry- Antibody-based techniques

Protein quantification techniques- Antibody based techniques - dye-binding techniques - Mass Spectrometry

Genome sequence databases

Protein sequence databases

Biological knowledgebases : - functions- pathways- seq. motifs- 3D structures

WORKFLOWS 1 : WORKFLOWS 1 : „classical“„classical“ 2D-PAGE 2D-PAGE

++ MALDI TOF MALDI TOF

11 22

3A3A 3B 3B 445 667 788

9910101111 1212

1

14

8

15

Workflow 1 : adaptation of bacteria to growth conditionsWorkflow 1 : adaptation of bacteria to growth conditions

Normal medium Low Glucose

Wick LM, et al, Environ Microbiol 3: 588-599, 2001

E.Coli adapts to a very low glucose medium by up- and downregulating a set of 15 proteins

M1

M2

M3

M4

M5

M6

M7

M8

Peptide Mass FingerprintingSearch with mass list

M1......M8

Tryptic digestion

Nr mw* pI* Acc. N. Name Function

1 52.2 5.07 P25553 aldA central metabolism

2 60.3 6.21 P23847 dppA peptide transport

3 47.5 5.06 P05313 aceA Central metabolism

4 48.45 6.71 P10904 ugpB transport

5 55.2 6.02 P00822 atpA proton transport/energy

6 ? ? P76108 ydcS transport

7 41.3 7.03 P02917 livJ amino acid transport

8 40.7 5.22 P02928 malE sugar transport

9 33.36 5..25 P02927 mglB sugar transport

10 61.5 5.81 P37192 gatY catabolism

11 30.9 7.76 P02925 rbsB sugar transport

12 25.8 5.22 P09551 argT amino acid transport

Workflow 1 : adaptation of bacteria to growth conditionsWorkflow 1 : adaptation of bacteria to growth conditions

Metabolic enzymes and transport proteins affected

Utilisation of alternative sources of energy

WORKFLOW 1 : quantitation and kineticsWORKFLOW 1 : quantitation and kinetics

UgpB (4)

00.020.040.060.080.100.12

1 2 3 4 5

DppA (2)

00.51.01.52.02.53.03.54.0

1 2 3 4 5

AldA (1)

00.20.40.60.81.01.21.41.6

1 2 3 4 5

AceA (3A)

0123456

1 2 3 4 5

AceA (3B)

00.20.40.60.81.01.2

1 2 3 4 5AtpA (5)

00.050.100.150.200.250.300.35

1 2 3 4 5

YdcS (6)

00.020.040.060.080.100.120.140.16

1 2 3 4 5

LivJ (7)

00.51.01.52.02.53.03.54.04.55.0

1 2 3 4 5MalE (8)

02468

101214

1 2 3 4 5

GatY (10)

00.51.01.52.02.53.0

1 2 3 4 5

RbsB (11)

00.10.20.30.40.50.60.70.80.9

1 2 3 4 5

MglB (9)

0246810

12141618

1 2 3 4 5

ArgT (12)

00.51.01.52.02.53.0

1 2 3 4 5

MalI (13)

00.050.100.150.200.25

1 2 3 4 5

Relative protein quantities as a function of time during the adaptation process

Time points : 1) Start – batch

culture2) Adapted

bacteria put back into high-glucose batch culture

3) 40 hr adaptation culture

4) 156 hr adaptation culture

5) 500 hr adaptation culture

• Ideal interface to biology• Analytical and micropreparative• Robust• Solid phase chemistry of proteins• Easy, low-tech• Removal of contaminants :

– At the loading point– After migration during fix / staining steps

Why is SDS-PAGE such a good preparation method?Why is SDS-PAGE such a good preparation method?

• protein digestion in gel: non quantitative• peptide sequence recovery: usually incomplete• whole protein recovery: poor

Disadvantages

In-gel digestion: solid phase chemistry of proteinsIn-gel digestion: solid phase chemistry of proteins

WORKFLOWS 2 : WORKFLOWS 2 :

General shotgun protein identification General shotgun protein identification techniquestechniques

example : example : Affinity pull-downAffinity pull-down

++ 1D-PAGE 1D-PAGE

++ LC-MS/MS LC-MS/MS

Shotgun sequencing from complex mixturesShotgun sequencing from complex mixtures

Denaturation, Proteolytic digestion

Db search

Multiprotein complex

List of identified proteins

1. P452182. P215433. Q125884. P326515. Q012456. ….

Complex peptide mixture(1000-20000 species)

Rp-LC-MSMS run

Alternative : MuDPIT (Multi Dimensional Protein Identification Alternative : MuDPIT (Multi Dimensional Protein Identification Technology) Technology)

Denaturation, Proteolytic digestion

Strong Cation Exchange(SCX)separation

Db search

Multiprotein mixture (complex)

List of identified proteins

1. P452182. P215433. Q125884. P326515. Q012456. ….

Complex peptide mixture(1000-20000 species)

Rp-LC-MSMS runs

Post-digestion separation of peptides by two-dimensional liquid chromatography Post-digestion separation of peptides by two-dimensional liquid chromatography instead of separation of proteinsinstead of separation of proteins

TIC: from 151002_ACO_B4strep.wiff Max. 5.3e5 cps.

5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 105 110 115Time, min

0.0

5.0e4

1.0e5

1.5e5

2.0e5

2.5e5

3.0e5

3.5e5

4.0e5

4.5e5

5.0e5

5.3e5

Inte

ns

ity, c

ps

653.36

133.06

526.26

406.23303.13 852.44

203.11199.19402.54186.12

472.25541.11

86.10536.34 449.15574.661064.42 629.30

665.38494.25

330.18 527.26

615.4186.10563.30

599.32

409.18

A VERY complex mixture – direct analysis (no separation)A VERY complex mixture – direct analysis (no separation)

Mascot Search ResultsUser : MQ Email : Search title :151002_ACO_B4strep.wiff : Angelos frac B4 IP strept MS data file : C:\DOCUME~1\paf\LOCALS~1\Temp\mas5D.tmp Database : Sprot 4028 (114033 sequences; 41888693 residues) Taxonomy : Mammalia (mammals) (23838 sequences) Timestamp : 17 Oct 2002 at 08:09:30 GMT

Significant hits:

ALBU_BOVIN (P02769) Serum albumin precursor (Allergen Bos d 6). DNM1_HUMAN (P26358) DNA (cytosine-5)-methyltransferase 1 (EC 2.1.1.37) AC15_HUMAN (P35251) Activator 1 140 kDa subunit (Replication factor C IF16_HUMAN (Q16666) Gamma-interferon-inducible protein Ifi-16 (Interfe K1CJ_HUMAN (P13645) Keratin, type I cytoskeletal 10 (Cytokeratin 10) ( K22E_HUMAN (P35908) Keratin, type II cytoskeletal 2 epidermal (Cytoker ACF7_HUMAN (Q9UPN3) Actin cross-linking family protein 7 (Macrophin) ( AC14_HUMAN (P35250) Activator 1 40 kDa subunit (Replication factor C 4 ALBU_FELCA (P49064) Serum albumin precursor (Allergen Fel d 2). AC15_MOUSE (P35601) Activator 1 140 kDa subunit (Replication factor C DYHC_MOUSE (Q9JHU4) Dynein heavy chain, cytosolic (DYHC) (Cytoplasmic AC12_HUMAN (P35249) Activator 1 37 kDa subunit (Replication factor C 3 EF11_CRIGR (P20001) Elongation factor 1-alpha 1 (EF-1-alpha-1) (Elonga RYR3_HUMAN (Q15413) Ryanodine receptor 3 (Brain-type ryanodine recepto K2C1_HUMAN (P04264) Keratin, type II cytoskeletal 1 (Cytokeratin 1) (K PLE1_RAT (P30427) Plectin 1 (PLTN) (PCN). AHNK_HUMAN (Q09666) Neuroblast differentiation associated protein AHNA TRYP_PIG (P00761) Trypsin precursor (EC 3.4.21.4). ACF7_MOUSE (Q9QXZ0) Actin cross-linking family protein 7 (Microtubule CENF_HUMAN (P49454) CENP-F kinetochore protein (Centromere protein F) ALBU_HUMAN (P02768) Serum albumin precursor. PLE1_HUMAN (Q15149) Plectin 1 (PLTN) (PCN) (Hemidesmosomal protein 1) TRI4_HUMAN (Q15650) Activating signal cointegrator 1 (ASC-1) (Thyroid NF1_HUMAN (P21359) Neurofibromin (Neurofibromatosis-related protein N NEBU_HUMAN (P20929) Nebulin

. . .

A VERY complex mixture still gives results, but...A VERY complex mixture still gives results, but...

450 500 550 600 650 700 750 800 850 900

m/z, amu

01020

40

60

80

100

120

140

160

180

Intensity, counts

18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48

Time, min

0.02.0e44.0e46.0e48.0e41.0e51.2e51.4e51.6e51.8e52.0e52.2e52.4e52.6e52.7e5

Intensity, cps

120.08157.15

545.75581.28545.75

652.36216.11681.35

670.84131.09 343.18498.55 738.39555.22 153.08561.29569.27 548.74 499.72489.53 445.07 499.70 469.25469.24 651.85

445.10 445.09 648.38

........ how deep are we going ?........ how deep are we going ?

analyzedmissed

some protein prefractionation

is necessary !

TNF family of ligands and TNF-receptor familyTNF family of ligands and TNF-receptor family

Bodmer JL et al, TIBS. 2002 27(1):19-26

(-)FasL

(+)FasL

FADD

Casp.8

Fas

Analysis of apoptotic signalling complexesAnalysis of apoptotic signalling complexes

Real lifeModel

The Fas (CD95) signalling complex (DISC)

Flip

Casp.10

Fc-FasL

?

?

Western blot

(-)BAFF

(+)FasL

Analysis of apoptotic signalling complexes : negative controlAnalysis of apoptotic signalling complexes : negative control

* run 1 cm* no fix, no stain !* cut, digest

1 cm

(-)BAFF

(+)FasL

LCMS runs Fas/Baff

TIC: from 180702BaffHL.wiff Max. 3.8e5 cps.

5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 105 110 115Time, min

0.0

2.0e4

4.0e4

6.0e4

8.0e4

1.0e5

1.2e5

1.4e5

1.6e5

1.8e5

2.0e5

2.2e5

2.4e5

2.6e5

2.8e5

3.0e5

3.2e5

3.4e5

3.6e5

3.8e5

Inte

ns

ity, c

ps

TIC: from FAS_180702_HL.wiff Max. 4.0e5 cps.

5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 105 110 115Time, min

0.0

2.0e4

4.0e4

6.0e4

8.0e4

1.0e5

1.2e5

1.4e5

1.6e5

1.8e5

2.0e5

2.2e5

2.4e5

2.6e5

2.8e5

3.0e5

3.2e5

3.4e5

3.6e5

3.8e5

4.0e5

Inte

ns

ity, c

ps

603.33

655.82

328.19

130.11

405.33

337.17

864.45

171.17582.29

643.87 603.69

158.06186.16

171.18 607.38120.11620.67449.15

BAFF complexHL sample

FasL complexHL sample

600 spectra

680 spectra

LC-MS/MS ANALYSISLC-MS/MS ANALYSISBAFF HL search resultsDatabase : MSDB 200402 (851746 sequences; 265326103 residues)Taxonomy : Mammalia (mammals) (166849 sequences)

Significant hits: Q96RF8 SSA1.- Homo sapiens (Human). BAB27292 AK010960 NID: - Mus musculus K2C1_HUMAN Keratin, type II cytoskeletal 1 (Cytokeratin 1) A45935 dnaK-type molecular chaperone hsc70 - mouse KRHU0 keratin 10, type I, cytoskeletal - human C25437 tubulin beta-3 chain - mouse Q9CWA2 ATP SYNTHASE, H+ TRANSPORTING MITOCHONDRIAL F1 CAA30026 HSHA44G NID: - Homo sapiens CAB59134 SEQUENCE 1 FROM PATENT WO9818921 PRECURSOR A44861 keratin, 67K type II epidermal - human Q9BWB7 HEAT SHOCK 70KD PROTEIN 9B (MORTALIN-) (Human). A26168 ribophorin I precursor - human PT0207 Ig gamma chain C region - chimpanzee PWHUA H+-transporting two-sector ATPase alpha chain precursor - human JC1473 H+-transporting ATP synthase (EC 3.6.1.34) alpha chain - mouse I77403 tubulin alpha-1 chain - human AAA57233 MUSHP7A2 NID: - Mus musculus CAA82315 HSKERAT9 NID: - Homo sapiens A29904 keratin 5, type II, epidermal - human HHHU84 heat shock protein 90-beta [validated] - human AAB86467 IMMUNOGLOBULIN GAMMA HEAVY CHAIN .-(Human). B26168 ribophorin II precursor - human CAA34756 HSEF1AC NID: - Homo sapiens S21097 alpha-1-antitrypsin precursor - bovine S04652 Ca2+-transporting ATPase (EC 3.6.1.38) 2, - pig CAA41735 BTBSA NID: - Bos taurus Q9UK02 BIP PROTEIN (FRAGMENT).- Homo sapiens (Human). KRHUEA keratin 6a, type II - human A22224 actin alpha, vascular smooth muscle - mouse Q96GA6 UNKNOWN (PROTEIN FOR MGC:15420).- Homo sapiens (Human). Q8WZ42 TITIN.- Homo sapiens (Human). I84741 RNA helicase - mouse Q9TS10 78 KDA APAMIN BINDING PROTEIN.- Bos taurus (Bovine). AAH02690 BC002690 NID: - Homo sapiens I48385 RNA helicase TNZ2 - mouse Q96FZ6 HEAT SHOCK 60KD PROTEIN 1 (CHAPERONIN).(Human). AAA56753 HSU15637 NID: - Homo sapiens CAA58470 HSPXMP11 NID: - Homo sapiens JQ0028 cytokeratin 19 – mouse…………………………………

FasL HL search results

Database : MSDB 200402 (851746 sequences; 265326103 residues)Taxonomy : Mammalia (mammals) (166849 sequences)

Significant hits: A37241 52K autoantigen Ro/SS-A - human Q8WUC1 TUBULIN, BETA 5.- Homo sapiens (Human). C25437 tubulin beta-3 chain - mouse CAC39526 SEQUENCE 15 FROM PATENT WO0129232.-HUman AAH19046 SIMILAR TO IMMUNOGLOBULIN HEAVY CONSTANT GAMMA 3 K1CJ_HUMAN Keratin, type I cytoskeletal 10 (Cytokeratin 10-(Human). I37383 FAS soluble protein - human A24903 tubulin alpha-1 chain - Chinese hamster A44861 keratin, 67K type II epidermal - human I38707 Fas ligand - human AAG41947 AF304164 NID: - Homo sapiens Q9BDN1 CD95L PROTEIN.- Cercocebus torquatus atys CAA82315 HSKERAT9 NID: - Homo sapiens AAB86467 IMMUNOGLOBULIN GAMMA HEAVY CHAIN (Human). NUCL_HUMAN Nucleolin (Protein C23).- (Human). 1ATS heat shock cognate protein 70 kD (44 kD chaperone ATPase 1FC1A Ig gamma-1 chain C region (Fc fragment), chain A - human I61769 keratin 6d, type II - human (fragment) A29904 keratin 5, type II, epidermal - human A40389 translation elongation factor eEF-1 alpha chain (clone pS1) - rat AAB46730 HSU86214 NID: - Homo sapiens CAD23746 IMMUNOGLOBULIN GAMMA HEAVY CHAIN CONSTANT REGION Q10466 TITIN, HEART ISOFORM N2-B (EC 2.7.1.-) (CONNECTIN).-(Human). Q8WZ42 TITIN.- Homo sapiens (Human). 1D3OA trypsin (EC 3.4.21.4), chain A - pig CAC20457 IMMUNOGLOBULIN HEAVY CHAIN CONSTANT GAMMA 4.- (Human). O75634 HEAT SHOCK PROTEIN 70 TESTIS VARIANT.- (Human). Q8WTZ6 RIBOSOMAL PROTEIN L18.- Homo sapiens (Human). 1NBMC f1-atpase (EC 3.6.1.34) delta and 1 epsilon subunits, chain C - bovine Q96PE2 TUMOR ENDOTHELIAL MARKER 4.- Homo sapiens (Human). Q9R1Q3 GLIAL FIBRILLARY ACIDIC PROTEIN ALPHA.- Rattus norvegicus (Rat). ITSH alpha-1-antitrypsin precursor - sheep CAA27396 MMACTBR2 NID: - Mus musculus Q8WXH0 NUANCE.- Homo sapiens (Human). Q9GL40 FAS ANTIGEN.- Macaca mulatta (Rhesus macaque). A33370 H+-transporting ATP synthase beta chain precursor, mitochondrial CAB76567 MMU250841 NID: - Mus musculus Q9GK28 FAS ANTIGEN APO-1/CD95.- Macaca arctoides (Stump-tailed macaque). Q9BZL4 MYOSIN BINDING SUBUNIT 85.- Homo sapiens (Human).…………………………………

Database search 1. Protein 1

2. Protein 23. Protein 34. …Database

search

Database search

1. Protein 12. Protein 23. Protein 34. …1. Protein 12. Protein 23. Protein 34. …

1. Protein 12. Protein 23. Protein 34. Protein 45. Protein 56. Protein 67. …

Total list sample 1

1. Protein 12. Protein 23. Protein 34. Protein 45. Protein 56. Protein 67. …

Total list sample 2

1. Protein 12. Protein 23. Protein 34. Protein 45. Protein 56. Protein 67. …

Total list sample 1

1) COMMON CONTAMINANTS ( PROTEINS STICKING TO BEADS)

2) LIGAND-COPURIFYING PROTEINS (EX SJOGREN SYNDROME 52 KDA)

3) COMMON HITS (WHAT IS IN BOTH LISTS)

Subtract from each list :

FILTERED RESULTSFILTERED RESULTS

FILTERED RESULTSFILTERED RESULTS

MASCOT DATABASE SEARCH Fas (CD95) complex

MASCOT DATABASE SEARCHBAFF receptor complex

ICE8_HUMAN (Q14790) Caspase-8

RO52_HUMAN (P19474) 52 kDa Ro protein (Sjogren syndrome type A antigen

RO52_HUMAN (P19474) 52 kDa Ro protein (Sjogren syndrome type A antigen

RS3_HUMAN (P23396) 40S ribosomal protein S3 RS3_HUMAN (P23396) 40S ribosomal protein S3

FADD_HUMAN (Q13158) FADD

K2C1_HUMAN (P04264) Keratin K2C1_HUMAN (P04264) Keratin

GC1_HUMAN (P01857) Ig gamma-1 chain C region GC1_HUMAN (P01857) Ig gamma-1 chain C region

CFLA_HUMAN (O15519) CASP8 and FADD-like apoptosis regulator

RS8_HUMAN (P09058) 40S ribosomal protein S8

GBLP_HUMAN (P25388) Guanine nucleotide-binding protein

GBLP_HUMAN (P25388) Guanine nucleotide-binding protein

TNR6_HUMAN (P25445) TNF-family receptor CD95

RL7A_MOUSE (P12970) 60S ribosomal protein L7a RL7A_MOUSE (P12970) 60S ribosomal protein L7a

RL7_HUMAN (P18124) 60S ribosomal protein L7 RL7_HUMAN (P18124) 60S ribosomal protein L7

ICEA_HUMAN (Q92851) Caspase-10

PHB_HUMAN (P35232) Prohibitin

K22E_HUMAN (P35908) Keratin K22E_HUMAN (P35908) Keratin

CLUS_HUMAN (P10909) Clusterin precursor CLUS_HUMAN (P10909) Clusterin precursor

RS3A_MOUSE (P97351) 40S ribosomal protein S3A RS3A_MOUSE (P97351) 40S ribosomal protein S3A

T13B_HUMAN (Q9Y275) Tumor necrosisfactor ligand superfamily member 13 (BAFF)

EF11_HUMAN (P04720) Elongation factor1-alpha 1 (EF-1-alpha-1) (Elonga

EF11_HUMAN (P04720) Elongation factor1-alpha 1 (EF-1-alpha-1) (Elonga

• One major evolution of proteomics technologies in the last years has been the introduction of gel-free approaches for large scale protein identification and quantification

• These methods combine isotope labelling, separation techniques and mass spectrometry

WORKFLOWS 3: WORKFLOWS 3: isotope labelling strategiesisotope labelling strategies

Relative quantification :

Comparison of proteins from samples A vs B ? Which proteins change in amount and how much ?

Applications :

-Healthy vs. diseased tissues

-Healthy vs. diseased body fluids

-Drug treated / untreated cells

-Stimulated / unstimulated cells

-Mutants / wt cells-……..

+TOF MS: Experiment 1, 44.071 to 46.012 min from 181203_QS_MQ_RuedaICAT1_long...a=3.56275471721098790e-004, t0=7.24150134716619500e+001

Max. 649.4 counts.

930 940 950 960 970 980 990 1000 1010 1020m/z, amu

0

20

40

60

80

100

120

140

160

180

200

220

240

260

280

300

320

Inte

ns

ity, c

ou

nts

8.96

968.52

9.96

1.00

10.962.00

m = 9 Da

Peptide from sample A

Peptide from sample B

Relative quantitation : stable isotope labelling is very fashionable!

Sample A : light isotope Sample B : heavy isotope

mix, digest

Quantitate and identify ( MS)

How to label ?

-chemically, post protein synthesis

“specific” chemical modification of AA side chain

(+) any sample can be done(-) side reactions

-metabolically, during protein synthesis

Incorporation of one or more labelled amino acid(+) “native” proteins(-) need cultivable organism

d0- or d8-ICATd0- or d8-ICAT

Biotin Biotin tagtag

Linker (heavy or Linker (heavy or light)light)

SS

NN NNOO

NN OOOO

OO NNII

OO OOXX

XX

XX

XX

XX

XX

XX

XX

ThiolThiolreactivereactive

(X= H or D)

State 1 State 2Transition states

[protein1][protein2]

.

.[proteinn]

[protein1][protein2]

.

.[proteinn]

Isotope Coded Affinity Tag (ICAT) reagents

Cell State 1 Cell State 2

Combine samples

Inte

nsity

m/z

A1

A2A3B1

B2Identify proteins by MS/MS

Inte

nsity

m/z

aa1aa2aa3

aa4

Quantitate protein levels by H8 / D8 peak heigth ratios

New Methods :New Methods : ICAT:quantitation ICAT:quantitation and identificationand identification

Biotin NH

O IHH

HH HH

HHBiotin N

H

O IDD

DD DD

DD

Modify with (H8)-ICAT

Modify with (d8)-ICAT

•Digest Trypsin•Purify Cys-containing peptides on avidin column

HS- -SH

Avidin Affinity Chromatography

control treatment

ICAT labelICAT label

Combine & proteolyzeCombine & proteolyze

d0d0

d0d0/d8d8

d8d8

Fraction # (Time (min))Fraction # (Time (min))

KC

l K

Cl

[M]

[M]

0.00.00.10.10.20.20.30.30.40.40.50.5

0.00.0

0.30.3

0.60.6

70701010 2020 3030 4040 5050 6060

Ion-Exchange

OD

OD

22 1414

%

%

AcN

AcNR

.A.

R.A

. (%

)(%

)

00

5050

100100

00

100100

5050

Time (min)Time (min)1010 2020 4040 5050 6060 70703030

LC-MS/MS

m/zm/z400400 800800 12001200 16001600 20002000

00

5050

100100 y9 y12y4 y8y5 y11 y14 y15 y17y13 y16y7y6 y10

b15b4 b10 b14b7 b8b6b3b2 b5 b9 b11 b12 b13R.A

. R

.A.

(%)

(%) D SQTNI N I AL T D A

A SLTAD NIQ NT DI

Area = 1.21x109

Area = 1.01x109

d0d0

d8d8 d8d8//d0d0 = 1:0.83

A) Identify

B) Quantify

1)

2)

3)

4)

5)

6)

Pair wise ICAT with Multidimensional Chromatography

ICAT (+) and (-)

- relative protein quantification by MS

- simplification of complex mixtures by selecting a subset of peptides after digestion

- eliminate analytical variability by mixing samples

~15 different isotope labelling methods developed in the last 5 years !!

- protein quantification unreliable for weak signals

- affinity purification (avidin) : losses for low amounts

- multiple side reactions possible

+

-

Recent ICAT studies (R. Aebersold’s group)Wollscheid B, von Haller PD, Yi E, Donohoe S, Vaughn K, Keller A, Nesvizhskii AI, Eng J, Li XJ, Goodlett DR, Aebersold

R, Watts JD.Lipid raft proteins and their identification in T lymphocytes.Subcell Biochem. 2004;37:121-52

Yan W, Lee H, Yi EC, Reiss D, Shannon P, Kwieciszewski BK, Coito C, Li XJ, Keller A, Eng J, Galitski T, Goodlett DR, Aebersold R, Katze MG.

System-based proteomic analysis of the interferon response in human liver cells .Genome Biol. 2004;5(8):R54.

Giglia-Mari G, Coin F, Ranish JA, Hoogstraten D, Theil A, Wijgers N, Jaspers NG, Raams A, Argentini M, van der Spek PJ, Botta E, Stefanini M, Egly JM, Aebersold R, Hoeijmakers JH, Vermeulen W.

A new, tenth subunit of TFIIH is responsible for the DNA repair syndrome trichothiodystrophy group A.Nat Genet. 2004 Jul;36(7):714-9.

Ranish JA, Hahn S, Lu Y, Yi EC, Li XJ, Eng J, Aebersold R.Identification of TFB5, a new component of general transcription and DNA repair factor IIH.Nat Genet. 2004 Jul;36(7):707-13.

Hardwidge PR, Rodriguez-Escudero I, Goode D, Donohoe S, Eng J, Goodlett DR, Aebersold R, Finlay BBProteomic analysis of the intestinal epithelial cell response to enteropathogenic Escherichia coli.J Biol Chem. 2004 May 7;279(19):20127-36.

Zhang J, Goodlett DR, Peskind ER, Quinn JF, Zhou Y, Wang Q, Pan C, Yi E, Eng J, Aebersold RH, Montine TJ. Quantitative proteomic analysis of age-related changes in human cerebrospinal fluid.

Neurobiol Aging. 2005 Feb;26(2):207-27.

Marelli M, Smith JJ, Jung S, Yi E, Nesvizhskii AI, Christmas RH, Saleem RA, Tam YY, Fagarasanu A, Goodlett DR, Aebersold R, Rachubinski RA, Aitchison JD.

Quantitative mass spectrometry reveals a role for the GTPase Rho1p in actin organization on the peroxisome membrane.

J Cell Biol. 2004 Dec 20;167(6):1099-112. Epub 2004 Dec 13.

• Label light / heavy cultures(Leu d0 / d3)

• Stimulate heavy cells

• Mix cells or lysates

• Purify fraction of interest

• Analyse by LC-MS/MS (->ID)

• Quantify signals of ion pairs

SILAC

Ong SE, Blagoev B, Kratchmarova I, Kristensen DB, Steen H, Pandey A, Mann M.

Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics.

Mol Cell Proteomics. 2002 May;1(5):376-86.

SILAC (+) and (-)

• relative protein quantification by MS

• eliminate praparative variability by mixing samples immediately after culture

• eliminate analytical variability

• peptides in native state (no side reactions)

• protein quantification unreliable for very weak signals

• mass shift variable (dependent on number of residues)

• only feasible with organisms in culture

+

-

Recent SILAC articles

Ong SE, Blagoev B, Kratchmarova I, Kristensen DB, Steen H, Pandey A, Mann M.Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics.Mol Cell Proteomics. 2002 May;1(5):376-86.

Blagoev B, Ong SE, Kratchmarova I, Mann M. Temporal analysis of phosphotyrosine-dependent signaling networks by quantitative proteomics.Nat Biotechnol. 2004 Sep;22(9):1139-45. Epub 2004 Aug 15.

Gruhler A, Olsen JV, Mohammed S, Mortensen P, Faergeman NJ, Mann M, Jensen ON. Quantitative Phosphoproteomics Applied to the Yeast Pheromone Signaling Pathway.Mol Cell Proteomics. 2005 Mar;4(3):310-327.

de Hoog CL, Foster LJ, Mann M.RNA and RNA binding proteins participate in early stages of cell spreading through spreading initiation centers.Cell. 2004 May 28;117(5):649-62.

Ong SE, Kratchmarova I, Mann M. Properties of 13C-substituted arginine in stable isotope labeling by amino acids in cell culture (SILAC).J Proteome Res. 2003 Mar-Apr;2(2):173-81.

Blagoev B, Kratchmarova I, Ong SE, Nielsen M, Foster LJ, Mann M.A proteomics strategy to elucidate functional protein-protein interactions applied to EGF signaling.Nat Biotechnol. 2003 Mar;21(3):315-8. Epub 2003 Feb 10.

Foster LJ, De Hoog CL, Mann M. Unbiased quantitative proteomics of lipid rafts reveals high specificity for signaling factors.Proc Natl Acad Sci U S A. 2003 May 13;100(10):5813-8. Epub 2003 Apr 30.

Other fields

• Proteome subsets– Phosphoproteome – Ubiquitinated proteins– …

• Clinical proteomics (marker discovery)– Too vast to summarise

• Proteome imaging– MALDI of tissues