ProteoRed Multicentric Experiment 5: Intensity-based Label-free quantification results

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

2. 1 mg 1 mg 1 mg 1 mg ProteoRed ME-5: Sample Laboratories that have used the intensity-based label-free approach have analysed the “old” sample

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. Sample preparation Protein denaturation with RapiGest (Waters) Standard tryptic digestion Direct loading onto the trapping column LC UPV/EHU: - nanoAcquity UPLC System (Waters) - 1.5 mg/run (3 replicates) - 120 min linear gradient (0-40% ACN) - Analytical column: BEH, 1.7mm, 200mm CIC bioGUNE: - nanoAcquity UPLC System (Waters) - 0.5 mg/run (5 replicates) - 90 min linear gradient (0-40% ACN) - Analytical column: BEH, 1.7mm, 100mm MSE 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 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: experimental conditions

6. ProteoRed ME-5: results Protein identifications

7. ProteoRed ME-5: results Peptide identifications Identified peptides: average number of peptides per run ( ): number of unique peptides identified with high confidence Average number of unique and high confidence peptides per protein

8. ProteoRed ME-5: results Protein quantification 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 UPV/EHU 153 CIC bioGUNE 140 104 ~ 70% 36 49 ~70% of the relatively quantified proteins are quantified in both laboratories

9. ProteoRed ME-5: results Quantification of spiked proteins * 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? CIC bioGUNE UPV/EHU Identified in ≥3 replicates with high confidence:140 proteins Absolutely quantified in ≥3 replicates: 153 proteins p<0.05 or p>0.95 ratio filtering: - variable among experiments - PME-5 > ±1.20 p<0.05: 11 proteins ratio filtering: - in general >± 1.25 - 1.5 - PME-5 > ±1.10 7 proteins (4 spiked + 3 E. coli) differentially expressed 5 (4 spiked + 1 E. coli) proteins differentially expressed 1 false positive quantification: 0.7% 0 false negative quantification 3 false positive quantification: 2.1% 0 false negative quantification

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