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Proteored Multicentric Experiment 8 (PME8)

Proteored Multicentric Experiment 8 (PME8) Quantitative Targeted Analysis in Proteomics. An Assesment Study (QTAPAS). ProteoRed WG1-WG2 Meeting Pamplona, December 10th 2013. Total 27 Participant Laboratories ( February - April 2013). 20 SRM data sets. 10 PRM data sets:.

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Proteored Multicentric Experiment 8 (PME8)

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  1. ProteoredMulticentric Experiment 8 (PME8) QuantitativeTargetedAnalysis in Proteomics. AnAssesmentStudy (QTAPAS) ProteoRed WG1-WG2 Meeting Pamplona, December 10th 2013

  2. Total 27 ParticipantLaboratories (February- April 2013) 20 SRM data sets 10 PRM data sets: 3 QTOF 7 OT

  3. SIGMA-ALDRICH MSQC1 • 6 Digestedproteins : • 25-fold concentrationrange – 3 concentrationtiers • Quantified UV beforedigestion • 2-3 labeledpeptides/ protein • L/H ratios 0.2-50 • Quantified AAA

  4. SAMPLE SET YeastDigest + Spiked MSQC1, 5 differentconcentrations fmol/ microgramYeastLysateDigest

  5. LABELED PEPTIDES

  6. QTAPAS - PME8 SRM ANALYSIS RECOMMENDED GUIDELINES • 1- LC conditions: • Nano LC- system: • Column: 75 mm x 15 cm C18 • Sample load: 1 mg yeast digest/injection • Flow rate: 300-500 nL /min • Gradient: 0-35% Acetonitrile in 90 min 3- Analysis design: - Samples A through E Replica 1 (from most diluted to most concentrated) - Blank runs (enough to secure clean baseline) - Samples A through E Replica 2 - Blank runs - Samples A through E Replica 3

  7. http://www.proteored.org/PME8_main.asp

  8. LINEARITY OF RESPONSE Average of 30 datasets – Error bars: StdDev. Tier 1

  9. Tier 2

  10. Tier 3

  11. ABSOLUTE QUANTIFICATION INTER LABORATORY VARIABILITY %CV 30 datasets Average of 30 datasets , three replicas averaged

  12. ABSOLUTE QUANTIFICATION INTER LABORATORY VARIABILITY N=20 N=7

  13. ABSOLUTE QUANTIFICATION INTER LABORATORY VARIABILITY N=20 N=3

  14. fmolmeasured

  15. %CV

  16. ABSOLUTE QUANTIFICATION Comparisonbytype of measurement

  17. ABSOLUTE QUANTIFICATION INTRA- vs INTER LABORATORY VARIABILITY Average, %CV 3 replicas - Average, %CV 30 datasets

  18. INTRA- vs INTER LABORATORY VARIABILITY Average, %CV 3 replicas - Average, %CV 30 datasets

  19. REPRODUCIBILITY OF TRANSITION PATTERN Transitionsignaldistribution Average 3 replicas Normalizeddotproducttoaveragedistribution

  20. REPRODUCIBILITY OF TRANSITION PATTERN

  21. INTRA- vs INTER LABORATORY VARIABILITY

  22. Analysis of pure MSQC1 (withoutyeastbackground) Forcomparison: Possibleeffects of complexbackground 3 Vials x 10 mg 200 nginjection – 80 fmol min

  23. EUPA 2013

  24. CONCLUSIONS • The results demonstrate a good degree of reproducibility of targeted quantification measurements by SRM at different laboratories, irrespective of the method of analysis and the spectrometer used. • The average Inter-Laboratory %CV of the measured absolute protein amounts ranges from less than 10%CV for Tier 1 proteins, to 40-60% for the proteins at the lowest concentrations. • The pattern of relative intensities of the transitions monitored is fairly consistent among different instruments and fragmentation modes, underscoring the utility of spectral databases for the design of quantification methods. • The results obtained at each laboratory allow the assessment of the limitations in sensitivity and limits of quantification under the diverse analytical conditions used

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