proteored wg1-wg2 meeting salamanca, march,16th 2010
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PME5: Quantitative LC-MS differential analysis F. Canals. ProteoRed WG1-WG2 Meeting Salamanca, March,16th 2010. THANK YOU!. Joan Josep Bech Núria Colomé Marta Monge. Salvador Martínez de Bartolomé Alex Campos … All participants in the study. LC-MS. => Isotopic labeling, Label free. - PowerPoint PPT PresentationTRANSCRIPT
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