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 > 1.5Geneva (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

22

1

4 laboratories3 laboratories2 laboratories

Proteins reported as differential

Labs proteins %123

71522979

64.520.77.1

4 44 4.05 19 1.76 22 2.0

UPF UPV BPG UCM PCBM PCBQ

Proteins 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 BP1 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 mlA1 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 C9 C10 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

IT Q-TOF TOF/TOF OT

0

2000

4000

6000

8000

10000

12000

14000

16000

Assigned MS2 Acquired MS2

MSMS Efficiency

IT # Acq. # Assign. % HCT-IT 8138 590 7.2LTQ-IT 8811 799 9.1HCT-IT 3417 445 13.0XCT-IT 2314 353 15.3

Q-TOF # Acq. # Assign. % Micro 203 101 49.8Q-Star 2639 1518 57.5Premier 2701Synapt 4296

TOF-TOF # Acq. # Assign. % 4800 1188 586 49.44800 1616 689 42.64800 773 503 65.14800 4513 3011 66.74700 979 414 42.3

OT # Acq. # Assign. % 8678 4143 47.76679 5769 86.4

13984 2166 15.54519 1902 42.1

0

50

100

150

200

250

300

350

Single Hit Proteins Total Id. Proteins

IT Q-TOF TOF/TOF OT

Protein Identification

0

50

100

150

200

250

300

350

Single Hit Proteins Total Id. Proteins

IT Q-TOF TOF/TOF OT

0

1000

2000

3000

4000

5000

6000

7000

8000

Total Unique Peptides

Protein Id

Peptide Id

Instrument

Number of E Coli proteins id. (total)

Total Number of proteins

quantitated

Number of proteins

quantitated > 3 peptides

Number of proteins

quantitated > 2 peptides

Number of proteins

quantitated 1 peptide

Not Quant

ICPL-CNB HCT-IT 109 105 37 36 32 4ICPL-HUVH HCT-IT 174 66 17 23 26 108

ITRAQ-CSIC-UAB LTQ-IT 209 209 122 36 51 0LF-UA XCT-IT 71 71 40 17 14 0

ITRAQ-CIPF QSTAR 125 111 54 65 25 14LF-CIC-BIOGUNE QTOF Premier 163 136 0 27

LF-UPV-EHU Synapt 313 312 304 312 0 1

ITRAQ-CNB1 4800 230 230 81 59 166 0ITRAQ-CNB2 4800 209 207 92 60 55 2ITRAQ-CNB3 4800 218 209 180 134 32 9

ITRAQ-ICS 4800 90 90 56 15 19 0ITRAQ-INIBIC 4800 129 128 34 53 42 1

ITRAQ UCM-PCM 4800 273 273 172 223 3 0ITRAQ MALDI-PCB 4700 44 32 25 7 0 12

ITRAQ Orbitrap PCB OT-Velos 163 139 119 20 0 24ITRAQ-UPF OT-Velos 313 199 52 64 83 114

LF-UB OT Velos 235TMT-direct R1 OT -Velos 275 194 125 153 0 81TMT direct R2 OT-Velos 290 191 131 156 1 99

TMT-Quixot OT-Velos 290 191 131 156 1 99

Quantification

0.000

0.200

0.400

0.600

0.800

1.000

1.200

1.400

1.600

1.800

ICPL ITRAQ TMT LF DIGE

Ratio variability

False positives ?

CYC_HORSE A/B=0.67

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.70

0.80

0.90

1.00

1.10

1.20

1.30

1.40

ICPL ITRAQ ITRAQ-CNB TMT LF DIGE

1000/1500 fmol / gQuantitation Results

MYG_HORSE A/B=2.60

0.00

1.00

2.00

3.00

4.00

5.00

6.00

ICPL ITRAQ ITRAQ-CNB TMT LF DIGE

520/200 fmol / gQuantitation Results

ALDOA_RABIT A/B=2.00

0.00

1.00

2.00

3.00

4.00

ICPL ITRAQ ITRAQ-CNB TMT LF DIGE

50/25 fmol / gQuantitation Results

ALB_BOVIN A/B=0.20

0.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40

1.60

1.80

ICPL ITRAQ ITRAQ-CNB TMT LF DIGE

5/1 fmol / gQuantitation Results

MYG_HORSE A/B=2.60

0.00

1.00

2.00

3.00

4.00

5.00

6.00

ICPL ITRAQ ITRAQ-CNB TMT LF DIGE

CYC_HORSE A/B=0.67

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.70

0.80

0.90

1.00

1.10

1.20

1.30

1.40

ICPL ITRAQ ITRAQ-CNB TMT LF DIGE

ALDOA_RABIT A/B=2.00

0.00

1.00

2.00

3.00

4.00

ICPL ITRAQ ITRAQ-CNB TMT LF DIGE

ALB_BOVIN A/B=0.20

0.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40

1.60

1.80

ICPL ITRAQ ITRAQ-CNB TMT LF DIGE

MRM

Conclusions

- Sample issues: Complexity of matrix, stability, solubility…

- Consistent quantitafication down to 25 fmol/ug. 1fmol level?

- Accuracy of “theoretical values”? SRM measurements

- CVs 10-20%. False differential protein Id.?

- Reporting data!