1. ProteoRed Multicentric Experiment 5: Intensity-based Label-free quantification results Kerman Aloria (University of the Basque Country, UPV/EHU) WG1-WG2 Meeting. Salamanca March, 16-17 th 2010

2. ProteoRed ME-5: Sample Laboratories that have used the intensity-based label-free approach have analysed the “old” sample  g

3. ProteoRed ME-5: methods and objectives based on the obtained results which proteins are differentially expressed in the sample?

4. ProteoRed ME-5: participants and approaches

5. ProteoRed ME-5: experimental conditions Sample preparation Protein denaturation with RapiGest (Waters) Standard tryptic digestion Direct loading onto the trapping column MS E acquisition - UPV/EHU: SYNAPT HDMS (Waters) - CIC bioGUNE: Q-Tof Premier (Waters) - Low collision energy scan: 1 s, 6 eV - High collision energy scan: 1 s, 12-35 eV LC UPV/EHU: - nanoAcquity UPLC System (Waters) - 1.5  g/run (3 replicates) - 120 min linear gradient (0-40% ACN) - Analytical column: BEH, 1.7  m, 200mm CIC bioGUNE: - nanoAcquity UPLC System (Waters) - 0.5  g/run (5 replicates) - 90 min linear gradient (0-40% ACN) - Analytical column: BEH, 1.7  m, 100mm Data processing and protein identification ProteinLynx Global Server Standard processing and searching parameters Relative quantification ProteinLynx Global Server UPV/EHU: Hi3 and relative quantification of estimated absolute quantifications CIC bioGUNE: Expression analysis based on comparative analysis of ion currents

6. ProteoRed ME-5: results UPV/EHUCIC bioGUNE Sample ASample BSample ASample B CYC_HORSE3 replicates 5 replicates MYG_HORSE3 replicates 5 replicates ALDOA_RABIT3 replicates 5 replicates ALBU_BOVIN3 replicates 5 replicates E. coli proteins219 (identified in ≥2 replicates) 160 (high confidence identifications) 164 (high confidence identifications) Protein identifications

7. ProteoRed ME-5: results UPV/EHUCIC bioGUNE Sample ASample BSample ASample B Identified peptides4296436325372701 peptide/protein18 1516 Peptide identifications UPV/EHU A1A2A3AverageB1B2B3Average CYC_HORSE42 (35)30 (30)35 (26)3047 (38)44 (38)42 (34)37 MYG_HORSE33 (26)46 (39)44 (35)3337 (32) 34 (30)31 ALDOA_RABIT22 (18)26 (23)31(27)2323 (19)21 (18)31 (14)17 ALBU_BOVIN19 (5)16 (3)16 (9)625 (15)27 (21)24 (14)17 ( ): number of unique peptides identified with high confidence Average number of unique and high confidence peptides per protein Identified peptides: average number of peptides per run

8. ProteoRed ME-5: results Relatively quantified proteins UPV/EHUCIC bioGUNE 153140 Protein quantification UPV/EHU 153 CIC bioGUNE 140 104 ~ 70% 49 36 Quantification criteria: - UPV/EHU: identified in 3 runs (out of 3) with at least 3 peptides - CIC bioGUNE: identified in 3 runs (out of 5) with high confidence ~70% of the relatively quantified proteins are quantified in both laboratories

9. ProteoRed ME-5: results Quantification of spiked proteins UPV/EHUCIC bioGUNE Theoretical ratio B/A Ratio B/A CV Sample A CV Sample B p (t-test)Ratio B/A Log(e) Ratio Log(e) SD* p* CYC_HORSE1.501.611.07.20.000831.490.400.031 MYG_HORSE0.380.417.99.50.000300.37-0.990.030 ALDOA_RABIT0.500.594.02.98.7 x 10 -5 0.83-0.190.040 ALBU_BOVIN5.02.0815.96.10.000751.430.360.191 * PLGS calculates log(e) SD and p values based on its own algorithms. p=1 indicates 100% of up-regulation and p=0 indicates 100% of down-regulation

10. ProteoRed ME-5: results Which proteins are differentially expressed between sample A and B? Ratio B/Ap value CYC_HORSE1.610.00083 MYG_HORSE0.410.00030 ALDOA_RABIT0.598.7 x 10 -5 ALBU_BOVIN2.080.00075 FETP_ECOLI0.202.4 x 10 -5 p 0.95 ratio filtering: - variable among experiments - PME-5 > ±1.20 Ratio B/Ap value CYC_HORSE1.491 MYG_HORSE0.370 ALDOA_RABIT0.830 ALBU_BOVIN1.431 PGM_ECOLI1.540.99 TRMJ_ECOLI1.380.98 ACKA_ECOLI0.770 7 proteins (4 spiked + 3 E. coli) differentially expressed 1 false positive quantification: 0.7% 0 false negative quantification p<0.05: 11 proteins Absolutely quantified in ≥3 replicates: 153 proteins UPV/EHU ratio filtering: - in general >± 1.25 - 1.5 - PME-5 > ±1.10 5 (4 spiked + 1 E. coli) proteins differentially expressed CIC bioGUNE Identified in ≥3 replicates with high confidence:140 proteins 3 false positive quantification: 2.1% 0 false negative quantification

11. ProteoRed ME-5: conclusions - MS E based acquisition can identify proteins within 3 orders of magnitude of dynamic range (sample A: ALBU_BOVIN 1 fmol/  g and CYC_HORSE 1000 fmol/  g) in complex samples - MS E based acquisition identifies on average >10 high confidence peptides per protein in complex samples Protein identification Protein quantification - MS E based label-free quantification can accurately measure relative protein ratios in complex samples - 70% (UPV/EHU) and 85% (CIC bioGUNE) of the identified proteins have been relatively quantified - Regularly used filtering criteria lead to a false positive quantification rate below 2% and 0 false negative quantifications. - ~70% of the quantified proteins are the same in both analysis MS E based acquisition combined with intensity-based label-free quantification have proven to be adequate tools for relative protein quantification in complex samples

12. Miren Josu Omaetxebarria Asier Fullaondo Jesus Mari Arizmendi Kerman Aloria Eva Rodríguez Suárez Felix Elortza