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

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

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

ProteoRed ME-5: participants and approaches

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, eV LC UPV/EHU: - nanoAcquity UPLC System (Waters)  g/run (3 replicates) min linear gradient (0-40% ACN) - Analytical column: BEH, 1.7  m, 200mm CIC bioGUNE: - nanoAcquity UPLC System (Waters)  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

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

ProteoRed ME-5: results UPV/EHUCIC bioGUNE Sample ASample BSample ASample B Identified peptides peptide/protein 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

ProteoRed ME-5: results Relatively quantified proteins UPV/EHUCIC bioGUNE Protein quantification UPV/EHU 153 CIC bioGUNE ~ 70% 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

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_HORSE MYG_HORSE ALDOA_RABIT x ALBU_BOVIN * 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

ProteoRed ME-5: results Which proteins are differentially expressed between sample A and B? Ratio B/Ap value CYC_HORSE MYG_HORSE ALDOA_RABIT x ALBU_BOVIN FETP_ECOLI x 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_ECOLI TRMJ_ECOLI ACKA_ECOLI 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 >± PME-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

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

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