Applicazioni di spettrometriadi massa in proteomica

Angela BachiDibit-San Raffaele Scientific Institute

Outline

• A SILAC approach for the global identification of endogenous PTMs

• Pathways analysis via label free expression proteomics

Small Ubiquitin-related Modifier: Sumo1

20 % identity with ubiquitin

Since 1996, more than 50 substrates involved in transcription, DNA repair, nuclear transport, signal transduction, cell cycle.

SUMOYLATION PATHWAY

• SUMO is synthesized as aprecursor and processed by hydrolases to make the carboxy-terminal double glycine motif available for conjugation.

• SUMO is activated in an ATP-dependent reaction by formation of a thioester bond with an E1 (SUMO-activating) enzyme.

• SUMO is transferred to the SUMO conjugating (E2) enzymeUbc9.

• A specific SUMO-E3 ligase enzyme might be required for efficient and properly modificationin vivo. (PIAS, PC2, RanBP2)

• The resulting isopeptide bond is stable and its disruption requires adesumoylating enzyme.

SC

O

Ubc9 SUMO

ΨKXESubstrate

HNC

O

SUMO

Ubc9

ATPAMP+PPi

SUMO SAE2SAE1

CS

O

E3ΨKXE

Substrate

NH2

SUMO

O

C O-

SAE2SAE1

HSE1

activating

E2conjugating

E3ligating

Biological functions of SUMO

chromatin structure

Antagonismof ubiquitination

protein/proteininteraction

SUMO

Substrate

Subnuclearlocalization

Nucleus-cytosolshuttling

Transcriptionfactor activity

Cell cycleRegulation

of DNA binding

MS characterization of the endogenously sumoylated proteins by aquantitative approach (SILAC)

SILAC (Stable Isotope Labeling by Amino acids in Cell culture)

[12C614N4-Arg] [13C6

15N4 -Arg]

Non stimulated cells MG132 treatmentStimulate cells6 plates of 8 doubling

1:1 Mix

Lysate in presence of 50mM NEM

Immunoprecipitation of SUMO-1 proteins using anti-SUMO antibody

beads

ProteinG

Anti SUMOIP

+

Quantitative Proteomics

SUMO-1 accumulates into nucleoli upon MG132 treatment

DMSO MG1321h

MG132 6h

MG13212h

a b c d

e f g h

i l m n

SUM

O-1

DN

AM

erge

Proteomic analysis of nucleolar sumoylated proteins

Hela Hela[12C6

14N4-Arg] [13C615N4 -Arg]

MG132 treatmentStimulate cells

Non stimulated cells

1:1 Mix

nucleoli purification

Lysate in presence of 50mM NEM

Immunoprecipitation of SUMO-1 proteins using anti-SUMO antibody

Tryptic digestion of gel bands and Identification by LC/MS-MS

Quantitative analysis : the MS and MSMS spectra are combined with Mascot results in a specific software: MaxQuant

SDS PAGE 10%Colloidal Coomassie

97

66

45

30

A1

B1

C1

D1

E1

F1

G1

H1

I1

L1

28-0

1-09

MW (kDa

)

M1

N1

O1

In gel digestion(Trypsin)

1C:\Xcalibur\data\09Sep\27Sep\AA1_7LM 9/28/2007 1:44:06 PM

RT: 8.95 - 77.50 SM: 7G

10 15 20 25 30 35 40 45 50 55 60 65 70 75Time (min)

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bund

ance

17.32

23.4929.43

38.9029.15 34.5626.55 33.50 35.4122.68

24.29 33.3631.64

20.71

16.52

37.6140.5616.27 41.53

15.5844.9310.53 47.19 62.61 70.8349.99 53.35

NL:8.43E4Base Peak F: ITMS + c NSI E d Full ms2 MS AA1_7LM

AA1_7LM #746 RT: 25.55 AV: 1 NL: 1.79E4T: ITMS + c NSI E d Full ms2 681.33@cid35.00 [175.00-1375.00]

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

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e Ab

unda

nce

963.2988

805.2812

672.2371

557.1674 876.2897

1116.1715399.0985718.2689486.1531 644.1760 1187.2200

539.1782371.1805300.0984 1003.1432246.2188 1062.3458589.1819440.1023 759.1253945.2876

858.3187215.0024 1227.2920 1300.2496

2

3 Protein identity

nLC-MS/MS

AutomationAutomationMass Scans Acquire:

• 1 high resolution scan for accurate mass on precursor ion and • 5 low resolution scans for MS/MS sequence information.

Add Mass to Exclusion List

Perform MS/MS with

Check Exclusion List

Select nth Most Abundant Ion

Full Scan MSMono isotopic accurate mass information of

peptides

Sequence informationof peptides

DataBase

Search

Parallel detection allows total cycle time of 1.0 sec.

1 sec

5 x 0.1 secin parallel

5 times

dta file

Requirements for large sets of protein ID data

• How often was the experiment repeated• Technical info on how data was created • Search parameters are adequately representing MS platform

• orbitrap-FT: 5 ppm precursor, 0.5 Da fragments• qTOF: 100 ppm precursor, 200 ppm fragments• Trap: 0.8 Da precursor, 0.8 Da fragments• Few variable modifications (ox M)

• Results based on MS/MS of tryptic peptides– Semi-tryptic peptides only allowed for most prominent fragmentation

pathway– Two or more independent tryptic peptides support ID– Alternatively multiple ID of the same peptide– ID must not rely on a single modified peptide

• Result representation should include– A score that relates to probability– Mass accuracy data– Peptide sequence including flanking amino acids– Count of how often a peptide is represented– Is the peptide match unique ?

MS characterization of the endogenously sumoylated proteins by aquantitative approach (SILAC)

Non stimulated cells MG132 treatment

Mix 1:1

IP, SDS-PAGE, LC-MS/MS

decreased sumoylation increased sumoylation

Plectin 1 isoform 10 (IPI00398778)

0.98 +- 0.07

R.LTVDEAVR.A K.GLVEDTLR.Q + 13C6-15N4 (R)

ATP-dependent RNA helicase (IPI00215638)

K.LAQFEPSQR.Q + 13C6-15N4 (R)

K.LAQFEPSQR.Q

0.70 +- 0.08

Putative sumoylated proteinsEntry Name ID MW (Da) Score Matched

peptides12C/13C+- S.D.

SUMO prediction sites

Subcellular localization

IPI00007928 Pre-mrna-processing-splicing Factor 8

273600 117 6 (2) 0.76+- 0.05

IKVE 0.94IKTE 0.94

Nucleolus

Cytosol

Cytosol

Nucleolus

Cytosol

Nucleolus

Nucleolus

Nucleus

Nucleolus

Nucleus

Nucleus

IPI00015953 Nucleolar RNA helicase 2 80121 454 15 (3) 0.62+-0.03

IKQD 0.94MKKE 0.80

Nucleolus

IPI00302592 Filamin A, Alpha. 280018 1850 50 (14) 0.82+- 0.07

VKAE 0.93VKVE 0.93

IPI00019502 Myosin-9. 227799 1798 53 (18) 0.82+- 0.13

VKND 0.93LKTD 0.91

IPI00420014 U5 Small Nuclear Ribonucleoprotein 200 Kda Helicase.

246032 337 12 (4) 0.75+- 0.09

VKLD 0.93VKYD 0.93

IPI00333015 Beta-spectrin 2 Isoform 2. 251948 365 13 (6) 0.87 +- 0.11

IKAE 0.94IKNE 0.94

IPI00005024 Myb-binding protein 1A 149731 1230 41 (12) 0.74+- 0.11

VKKD 0.93LKAD 0.91

IPI00215638 ATP-dependent RNA helicase A

142103 1250 39 (17) 0.79+- 0.08

IKSE 0.94LKNE 0.91

IPI00140420 EBNA-2 co-activator variant(Fragment)

108222 141 5 (2) 0.83+-0.01

IKCP 0.84IKNG 0.77

IPI00017451 Splicing factor 3 subunit 1 88888 70 4 (1) 1.03 LKKE 0.91LKTE 0.91

IPI00017297 Matrin-3 94921 137 5 (2) 0.81+-0.01

IKNE 0.94VKVD 0.93

IPI00449049 Poly-ADP ribose polymerase1

113680 393 11 (3) 0.87+- 0.02

IKDE 0.94VKAE 0.93

Putative sumoylated proteinsEntry Name ID PM Score Matched

peptides12C/13C+- S.D.

SUMO prediction sites

Subcellular location

IPI00015953 Nucleolar RNA helicase 2 80121 454 15 (3) 0.62+-0.03

IKQD 0.94MKKE 0.80

MKEE 0.80AKLD 0.79

AKFE 0.79

AKVE 0.79

VKLP 0.82LKAG 0.73

VKKG 0.76

IKED 0.94GKIE 0.67

MKEE 0.80

FKAF 0.50

AKSD 0.79GKHE 0.68

IPI00221106 Slicing factor 3B subunit 2 98169 156 7 (3) 0.77+- 0.06

VKKE 0.93LKIP 0.80

Nucleolus

IPI00021405 Lamin-A/C 74380 906 30 (12) 0.78+-0.08

Nucleolus

Nucleolus

Endoplasmic reticulum

Nucleolus

Nucleolus

Nucleolus

Nucleolus

Nucleolus

Nucleolus

IPI00003362 Bip protein 72492 357 12 (4) 0.62+-0.04

IPI00012772 60S ribosomal protein L8 28104 126 6 (2) 0.73+-0.04

IPI00021840 40S ribosomal protein S6 28834 120 3 (2) 0.64+-0.01

IPI00215965 heterogeneous nuclear ribonucleoprotein A1isoform b

38837 165 6 (3) 0.86+-0.02

IPI00553164 40S ribosomal protein SA 32816 295 8 (3) 0.74+-0.10

IPI00465361 60S ribosomal protein L13 24173 80 5 (1) 0.72+-0.01

IPI00025512 Heat-shock protein beta-1 22826 140 4 (1) 0.45+-0.06

Nucleolus

IPI00304596 Non-POU domain-containing octamer-binding protein

54232 300 13 (4) 0.76+-0.12

Results225 proteins identified (75 quantitatively affected)

Belong to: RNA metabolism, DNA binding, signalling

Potential SUMO targets because belong to family of known SUMO targetsRibosomal protein 18hnRNP 11Splicing factor 3DNA helicase 1RNA helicase 9Nuclear pore complex 2Transcription factor 2Elongation factor 3Histone 11Initiation factor 2Topoisomerase 1

11 Known SUMO targets:

GTP-binding proteinSWI/SNF proteinDNA topoisomerase IIActin binding proteinhnRNP KTubulinFact complexHistone H2AHistone H2BHistone H1Histone H4

ConclusionsThe described approach allowed the identification and quantitation of

several SUMO proteins involved mainly in:nucleic acid binding and regulation (RNA helicase, hnrp, snrp, splicing

factor,initiation and elongation factor, ribosomal protein), protein trafficking (importin)

For most potential sumoylated proteins the ratio is 0.6-0.8 with an average S.D. of 0.06 indicating that two-peptide pairs already lead to reasonably accurate quantification with rather small SD

Upon inhibition of proteasome it is evident an accumulation and a nucleolar redistribution of SUMO signal

We have validated some of the new SUMO target by in vitro reaction and WB

Pathways analysis in proteomics

the input is the expression proteomics data and the

output is the list of activated or dominant pathways in a given sample

Aim- To generate non-trivial functional hypotheses on

biological systems

- To define disease biomarker among pathways or pathway patterns instead of single molecules

- To rationalize how molecules interact in ‘molecular pathways’, i.e. chains of chemical reactions or physical interactions in which the product of one reaction becomes the reactant of the other.

MS-based techniques for quantitative proteomics

Stable Isotope Labelling

In vivo Labelling:• 14N/15N media

• SILAC

Suitable for mitotically active tissues and cell lines!

In vitro Labelling:• N-terminal peptide labelling (iTRAQ)

• C-terminal peptide labelling (16O/18O incorporation via proteolysis)

• Amino acid-based labelling (ICAT)

Suitable for mitotically active tissues and cell line, but also for resting cells such as neurons!

label-free quantitation

• mass spectrometry based approach

• less time consuming

• less sample consuming, if coupled to gel-free proteomic experiments

Advantages:

• no need for labelled amino acids

• can be applied to non-proliferating cells

Limitations:

• relatively new technique

• might not be as accurate as SILAC

Quantitation taking into account protein relative abundances:

• peptide score summation

• number of peptides identifying a protein

• use of a pseudo-internal standard

label-free quantitation

Abundance of the below peptide

MS of a peptide

Sum of isotopic peaks

RetentionTime

Accurate mass

Chromatographic peak

Differential Analysis of Promonocytic U937 “Plus”And “Minus” Cell Clones

Promonocytic U937 cell line is used as in vitro model of HIVinfection.

Two different clones of U937 have been described and defined as “Plus” and “Minus” in respect of their efficiency or inefficiency to support productive HIV-1 infection.

Aim of this study was to investigate the whole proteome of Plus (10) and Minus(34) clones in order to detect potential quali/quantitative differences at the protein expression level to unravel protein correlates of efficient/inefficient HIV replication.

Known differences at morphological, proliferative and molecular levels

Expression of common and differential myelomonocytic Ag in U937 Plus and

Minus cell clones

Cellular factors differentially expressed by Plus and Minus

U937 cell clones

Mass spectrometry flow chart

PROTEIN SAMPLE

REDUCTION + ALKYLATION

TRYPTIC DIGESTION

PEPTIDE MIXTURE

PEPTIDE SEPARATION BY

RP-nHPLC

MS AND MS/MS SPECTRA ACQUISITION

RAW DATA RETENTION TIME

PEAK INTENSITY

FRAGMENTATION PATTERN

DATABASE SEARCH WITH MASCOT

PROTEIN IDENTIFICATION

PEPTIDE ION SCORE

PROTEIN SCORE

NUMBER OF MATCHING PEPTIDES

C:\Xcalibur\...\10Oct\03Oct\UR_Mec_strip 10/3/2008 11:12:59 AM

RT: 0.00 - 94.01 SM: 7G

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90Time (min)

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bund

ance

17.69

20.64

23.96

28.28

15.6226.26

33.0659.5049.99 58.24 60.40

39.28 41.40 61.2135.66 44.41 56.79 65.2587.2014.48 90.9632.70 65.86

68.45 78.03 79.8310.296.12

NL:6.79E5Base Peak F: ITMS + c NSI E d Full ms2 MS UR_Mec_strip

UR_Mec_strip #695 RT: 17.92 AV: 1 NL: 4.31E3T: ITMS + c NSI E d Full ms2 766.86@cid37.00 [200.00-1545.00]

200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bund

ance

752.8802

634.2949

1004.4404899.2284

876.3281681.7055

547.2401986.2805

1132.5461598.1921 786.2758

1100.3735529.2611 1229.3463 1358.4312304.2217 433.2450 1048.4042833.7974 1261.4939

272.1626 1518.44311412.3185

OrbiTrap

•SINGLE-STEP ANALYSIS

About 250 ng of tryptic digest have been injected into the nLC and separated with a 194 minutes long gradient.

C:\Xcalibur\data\09Sep\27Sep\AA1_7LM 9/28/2007 1:44:06 PM

RT: 8.95 - 77.50 SM: 7G

10 15 20 25 30 35 40 45 50 55 60 65 70 75Time (min)

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e Ab

unda

nce

17.32

23.4929.43

38.9029.15 34.5626.55 33.50 35.4122.68

24.29 33.3631.64

20.71

16.52

37.6140.5616.27 41.53

15.5844.9310.53 47.19 62.61 70.8349.99 53.35

NL:8.43E4Base Peak F: ITMS + c NSI E d Full ms2 MS AA1_7LM

AA1_7LM #746 RT: 25.55 AV: 1 NL: 1.79E4T: ITMS + c NSI E d Full ms2 681.33@cid35.00 [175.00-1375.00]

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

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bund

ance

963.2988

805.2812

672.2371

557.1674 876.2897

1116.1715399.0985718.2689486.1531 644.1760 1187.2200

539.1782371.1805300.0984 1003.1432246.2188 1062.3458589.1819440.1023 759.1253945.2876

858.3187215.0024 1227.2920 1300.2496

MS high resolutionTop 5 low resolution

•SINGLE-STEP ANALYSIS

About 250 ng of tryptic digest have been injected into the nLC and separated with a 180 minutes long gradient.

Mass spectra have been acquired twice with two partially overlapping mass ranges:

•300-900 low mass

•750-1600 high mass

U937_10 U937_10bis U937_34 U937_34bis

12415 10689

Number of proteins (bold red and ion score more than 20)

664 733 752 680

1370810510Number of queries

Mass ranges combined. Mascot and X! tandem searches. 5ppm precursor, 0.5 Da for fragments. At least 2 peptides at 95%, protein probability >99%.

more than 700 proteins identified

Peptide QUANTIficationSEQUENCE RT MASCOT RT APEX MZ MASCOT MZ QUANTI IPI MASCOT SCORE INTENSITY FULLINTR.YESLTDPSKLDSGK.E 91.200607 84.407524 770.381287 770.379028 IPI00784295 61 212771.6406K.HLEINPDHSIIETLR.Q 86.204216 86.17421 893.976074 893.978271 IPI00784295 64 65058548K.VILHLKEDQTEYLEER.R 83.876076 83.714897 1008.028015 1008.027954 IPI00784295 70 10953614K.DLVILLYETALLSSGFSLEDPQTHANR.I 133.445953 131.745605 1001.520325 1001.524231 IPI00784295 137 203456512M.PEETQTQDQPMEEEEVETFAFQAEIAQLMSLIINTFYSNK.E 173.490768 173.519012 1560.401001 1560.397095 IPI00784295 54 182668.7188M.PEETQTQDQPMEEEEVETFAFQAEIAQLMSLIINTFYSNKEIFLR.E 177.030182 176.660629 1335.139771 1335.140381 IPI00784295 26 87048.85938K.TLNDELEIIEGMK.F 82.980316 77.69648 752.882202 752.881042 IPI00784154 96 1714931.5R.ALMLQGVDLLADAVAVTMGPK.G 125.89357 126.296585 1057.073608 1057.074341 IPI00784154 98 118192832R.TALLDAAGVASLLTTAEVVVTEIPK.E 201.519104 95.73555 828.140686 828.142761 IPI00784154 69 1631606.125K.VVIGMDVAASEFFR.S 122.195076 119.691261 770.89563 770.895142 IPI00465248 115 32162.14258K.IDKLMIEMDGTENK.S 105.149544 103.420265 818.901428 818.9021 IPI00465248 56 147749.8281R.AAVPSGASTGIYEALELR.D 92.408096 92.50663 902.976379 902.978149 IPI00465248 93 456041760K.LAMQEFMILPVGAANFR.E 101.599594 101.737404 954.499329 954.499756 IPI00465248 70 258961744K.FTASAGIQVVGDDLTVTNPK.R 112.769814 117.65992 1017.031128 1017.029846 IPI00465248 88 78492.90625K.FTASAGIQVVGDDLTVTNPKR.I 88.349304 88.265793 1095.084839 1095.081543 IPI00465248 111 9042372

Protein QUANTIfication

IPI AbundanceIPI00784295 279951163.2IPI00784154 121539369.6IPI00465248 724304280.9… …… …

Sum of the intensity of everypeak belonging to that protein:

SINGLE-STEP ANALYSIS

Total lysates in-solution digested: 250 ng of digested proteins separated by LC through very long gradient. Analysis in duplicates.

MS acquired two times, with two different and partially overlapping mass ranges (300-900 and 750-1600).

Mass spectra summed up and submitted to database searching as a whole; MASCOT and X! tandem algorithms for database search.

More than 700 proteins identified and quantitated :

19 unique of clone 10+

47 unique of clone 34-

64 up regulated and 27 down-regulated in 34

Go analysis

Use of known signalling mechanismsto identify and quantify activated pathways

What do we want:

Detecting events in unbiased way to: Hypothesisgeneration

PSE Zubarev et al. J Proteomics. 2008

Protein names and protein abundances are loaded

Two analysis can be performed: direct and TF mediated, but both pass through the Key Node (signaling molecules found on pathway intersections in the upstream vicinity of the genes from the input list) filtering step.

The resultant sets of genes are compared and their intersection is mapped on a pathway database.

Each found key-node receives a score reflecting its connectivity, i.e. how many input-list genes are reached and the proximities to the reached genes.

Key-nodes with the highest connectivity (highest score) are then selected, and downstream genes are chosen as a subset for subsequent mapping onto the pathways.

U937_34Long Vs U937_10LongTop 3

Outliers(> 3 σ)

Two dominant pathways in Minus clone : FAS and JNK, while IFN resulted dominant in the Plus clone

RESULTSUp to now, the analysis on the two clones showed differences at a morphological, proliferative and molecular level (such as the expression of surface antigens, proteases, and membrane receptors).

A new dimension has been added by pathways analysis.

..but needs to be validated

Involved pathways:Fas and JNK activated in U937 clone 34IFN upregulated in U937 clone 10

To confirm this hypothesis, different antibodies against elements of these pathways have been used in western blot analysis. In particular: anti-casp3 (cleaved form) (FAS pathway)anti-pJNK (active form) (JNK pathway)anti-CASP8 (FAS pathway)

Testing the hypothesis

#34 #34#10 #10

Fas pathway is activated in clone 34

Anti Caspase-8

The JNKs family includes; JNK1 (four isoforms), JNK2 (four isoforms), and JNK3 (two isoforms). JNKs are activated by MAP2kinases The activated JNK/SAPK translocate to the nucleus where they phosphorylate transcription factors such as c-Jun,Elk1,DPC4

JIP3JIP2

NUCLEUS

CYTOPLASM

MEMBRANE

MAPKKKs

JNK1/3

JIP1MKK4/7

JNKJNK

GTPaseGTPaseGTPase

Growth factors, UV, Inflammatory cutokines

Cellular stress, γ radiation

TF transcription

GrowthDifferentiationSurvivalApoptosis

Testing the hypothesis

JNK1 is activated in clone 34, while

JNK2/3 are activated in clone 10

47.5

32.5Actin

62

47.5 Anti pSAPK/JNK

#10#34 #34#10

expected MW: approx 46 kDA (pJNK1) and 54 kDa (pJNK2/3)

Where are we?

We can , in 1 exp. from 1x106 cells, identify and relatively quantitate (1 status vs the other) about 500-1000 proteins.

Pathway Search Engine

Dominant pathwayidentification

Eduardo Salce

Umberto Restuccia

Alfonsina D’Amato

Angela CattaneoVittoria Matafora

Vera Usuelli

Federico Torta