ProteoRed WG1-WG2 Meeting

Salamanca, March,16th 2010

PME5: Quantitative LC-MS differential analysis

F. Canals

Joan Josep BechNúria ColoméMarta Monge

THANK YOU!

Salvador Martínez de BartoloméAlex Campos…

All participants in the study

2008 MULTICENTRIC STUDY

4 + 4 technical replicates

Untreated Cells

2DE-DIGE

Sample 1

Treated Cells (+ EGF 24 h)

Sample 2

Image analysis:

- NLD- LUDESI- GE

Protein Identification:

30 top differential protein spots > 1.5 foldp < 0.05

LC-MS => Isotopic labeling, Label free

MDA-MB-468 Br.Cancer Cells

BPG-Geneve TMT Off-Gel MALDI TOF/TOF 4800PCB iTRAQ High pH RP MALDI TOF/TOF 4700PCB iTRAQ High pH RP ESI- QTOF Q-STARPCM iTRAQ SCX MALDI TOF/TOF 4800UPF iTRAQ SCX ESI- QTOF Q-STARUPV LF ESI- QTOF Synapt

PROTEORED ASSAY 2008

Gel-free quantitative approaches

PROTEORED ASSAY 2008

Identified proteins Quantified proteins Identified peptides Quantified peptides Protein ratio > 1.5

Geneva (Off-Gel) 412 179 812 1105 40PCB (RP high pH) 1005 286 1884 1584 26PCM-UC (SCX) 569 564 4861 4861 1UPF 312 (1441) 307 4907 2093 12UPV-EHU 297 (650) 274 1907 1907 19 + 23

11

1

124

1

2

2

1

4 laboratories

3 laboratories

2 laboratories

Proteins reported as differential

Labs proteins %1

2

3

715

229

79

64.5

20.7

7.14 44 4.05 19 1.76 22 2.0

UPF UPV BPG UCM PCBM PCBQ

Proteins Identified by LC/MS/MSProteins Identified by LC/MS/MS

“Centralized” analysis of data

“Collective” analysis of “pooled” data

Next Multicentric Experiment on (Quantitative) LC-MS experiment

1- Run LC-MS/MS of a sample of “medium” complexity in triplicate

-> evaluate reproducibility -> Different protocols – SOP ?

2- 2a) Two standard mixtures A, B:

Protein 1 50 fmol A1-B1 -> 1 : 5Protein 2 500 fmol A2-B2 -> 1 : 2Protein 3 5 pmol A3-B3 -> 1.5:1

2b) A, B + Matrix (medium complexity)

-> Run triplicates 2a, 2b ?

Matrix -> Bacterial lysate?, Fraction cell lysate (supplied?)SOP-> Include Fractionation ? Labeling – Label Free -> assign # labs?

LC gradient Search parameters

PME09

A B A/B fmol A fmol B Prot Mw ng A ng B

P1 1.0 1.5 0.67 1,000 1,500 Cyt C 12362 12.36 18.54

P2 2.6 1.0 2.60 520 200 Myo 16952 8.82 3.39

P3 2.0 1.0 2.00 50 25 Aldo A 39212 1.96 0.98

P4 1.0 5.0 0.20 1 5 BSA 66430 0.07 0.33

23.2 23.2

- Matrix: Bacteriophage T4 capside proteins: ~35 proteins- > adequate complexity for single LC-MS runs - > replicas

-Samples: A , B

-> 4 level spiked proteins – 3 orders of magnitude-> relative abundance 0.38 - 5

Per 1 g T4 proteins

• Distribute ~100 g each sample A,B

• Isotope label (i-TRAQ, ICPL, O18…) or label-free relative quantitation A/B

-> minimum of 4 replicas: evaluate variability, quantitation accuracy…

• 2DE-DIGE?

• Provide unified database (E.Coli + spiked proteins). Inter-Lab comparisons.

• Report using MIAPE document generator

PME5

E. Coli sample preparation

ProteoredQ 091009 Run 3001:1_UV1_215nm ProteoredQ 091009 Run 3001:1_UV2_280nm ProteoredQ 091009 Run 3001:1_Fractions ProteoredQ 091009 Run 3001:1_Logbook ProteoredQ 091009 Run 3001:1_A905Temp

0

500

1000

1500

2000

mAU

0.0 5.0 10.0 15.0 20.0 ml

A1 A2 A3 A4 A5 A6 A7 A8 A9 A10 A12 A14 B1 B2 B3 B4 B5 B6 B7 B8 B9 B10 B12 B14 C1 C2 C3 C4 C5 C6 C7 C8 C9C10 C12 Waste

A9 A10 A11 A13 A14 B4 B5 B7 B9 B11

SCX

10/40 fractions100-300 proteins Id

Off-gel fractionation: 902 prots

Cytoplasmatic fraction

- 4 DIGE

- 2 ICPL

- 11 iTRAQ

- 3 TMT

- 6 LF