1. Proteored Multicentric Experiment 8 (PME8) Quantitative Targeted Analysis in Proteomics. An Assesment Study (QTAPAS) ProteoRed WG1-WG2 Meeting Pamplona, December 10th 2013

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

5. SIGMA-ALDRICH MSQC1 - 6 Digested proteins : 25-fold concentration range – 3 concentration tiers Quantified UV before digestion - 2-3 labeled peptides/ protein L/H ratios 0.2-50 Quantified AAA

6. SAMPLE SET fmol / microgram Yeast Lysate Digest Yeast Digest + Spiked MSQC1, 5 different concentrations

7. LABELED PEPTIDES

8. 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 QTAPAS - PME8 SRM ANALYSIS RECOMMENDED GUIDELINES 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

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

10. LINEARITY OF RESPONSE Average of 30 datasets – Error bars: Std Dev. Tier 1

11. Tier 2

12. Tier 3

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

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

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

16. fmol measured

17. %CV

18. ABSOLUTE QUANTIFICATION Comparison by type of measurement

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

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

21. REPRODUCIBILITY OF TRANSITION PATTERN Transition signal distribution Average 3 replicas Normalized dot product to average distribution

22. REPRODUCIBILITY OF TRANSITION PATTERN

23. INTRA- vs INTER LABORATORY VARIABILITY

26. 200 ng injection – 80 fmol min Analysis of pure MSQC1 (without yeast background) For comparison: Possible effects of complex background 3 Vials x 10  g

27. EUPA 2013

28.  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 CONCLUSIONS