proteored multicentric experiment 5: intensity-based label-free quantification results
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WG1-WG2 Meeting. Salamanca March, 16-17 th 2010. ProteoRed Multicentric Experiment 5: Intensity-based Label-free quantification results Kerman Aloria (University of the Basque Country, UPV/EHU). 1 m g. 1 m g. 1 m g. 1 m g. ProteoRed ME-5: Sample. - PowerPoint PPT PresentationTRANSCRIPT
ProteoRed Multicentric Experiment 5:
Intensity-based Label-free quantification results
Kerman Aloria(University of the Basque Country, UPV/EHU)
WG1-WG2 Meeting. SalamancaMarch, 16-17th 2010
ProteoRed ME-5: Sample
Laboratories that have used the intensity-based label-free approach have analysed the “old” sample
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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 conditionsSample preparation
Protein denaturation with RapiGest (Waters)Standard tryptic digestion
Direct loading onto the trapping column
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
LC
UPV/EHU:- nanoAcquity UPLC System (Waters)- 1.5 g/run (3 replicates)- 120 min linear gradient (0-40% ACN)- Analytical column: BEH, 1.7m, 200mm
CIC bioGUNE:- nanoAcquity UPLC System (Waters)- 0.5 g/run (5 replicates)- 90 min linear gradient (0-40% ACN)- Analytical column: BEH, 1.7m, 100mm
Data processing and protein identificationProteinLynx Global Server
Standard processing and searching parameters
Relative quantificationProteinLynx 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/EHU CIC bioGUNE
Sample A Sample B Sample A Sample B
CYC_HORSE 3 replicates 3 replicates 5 replicates 5 replicates
MYG_HORSE 3 replicates 3 replicates 5 replicates 5 replicates
ALDOA_RABIT 3 replicates 3 replicates 5 replicates 5 replicates
ALBU_BOVIN 3 replicates 3 replicates 5 replicates 5 replicates
E. coli proteins 219 (identified in ≥2 replicates)
219 (identified in ≥2 replicates)
160 (high confidence identifications)
164 (high confidence identifications)
Protein identifications
ProteoRed ME-5: results
UPV/EHU CIC bioGUNE
Sample A Sample B Sample A Sample B
Identified peptides 4296 4363 2537 2701
peptide/protein 18 18 15 16
Peptide identifications
UPV/EHUA1 A2 A3 Average B1 B2 B3 Average
CYC_HORSE 42 (35) 30 (30) 35 (26) 30 47 (38) 44 (38) 42 (34) 37
MYG_HORSE 33 (26) 46 (39) 44 (35) 33 37 (32) 37 (32) 34 (30) 31
ALDOA_RABIT 22 (18) 26 (23) 31(27) 23 23 (19) 21 (18) 31 (14) 17
ALBU_BOVIN 19 (5) 16 (3) 16 (9) 6 25 (15) 27 (21) 24 (14) 17
( ): number of unique peptides identified with high confidenceAverage 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/EHU CIC bioGUNE
153 140
Protein quantification
UPV/EHU153
CIC bioGUNE140
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
ProteoRed ME-5: results
Quantification of spiked proteins
UPV/EHU CIC 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 1.50 1.61 1.0 7.2 0.00083 1.49 0.40 0.03 1
MYG_HORSE 0.38 0.41 7.9 9.5 0.00030 0.37 -0.99 0.03 0
ALDOA_RABIT 0.50 0.59 4.0 2.9 8.7 x 10-5 0.83 -0.19 0.04 0
ALBU_BOVIN 5.0 2.08 15.9 6.1 0.00075 1.43 0.36 0.19 1
* 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: resultsWhich proteins are differentially expressed between sample A and B?
Ratio B/A p value
CYC_HORSE 1.61 0.00083
MYG_HORSE 0.41 0.00030
ALDOA_RABIT 0.59 8.7 x 10-5
ALBU_BOVIN 2.08 0.00075
FETP_ECOLI 0.20 2.4 x 10-5
p<0.05 or p>0.95ratio filtering: - variable among experiments - PME-5 > ±1.20
Ratio B/A p value
CYC_HORSE 1.49 1
MYG_HORSE 0.37 0
ALDOA_RABIT 0.83 0
ALBU_BOVIN 1.43 1
PGM_ECOLI 1.54 0.99
TRMJ_ECOLI 1.38 0.98
ACKA_ECOLI 0.77 0
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 bioGUNEIdentified in ≥3 replicates with high confidence:140 proteins
3 false positive quantification: 2.1%0 false negative quantification
ProteoRed ME-5: conclusions
- MSE 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
- MSE based acquisition identifies on average >10 high confidence peptides per protein in complex samples
Protein identification
Protein quantification
- MSE 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
MSE based acquisition combined with intensity-based label-free quantification have proven to be adequate tools for relative protein quantification in
complex samples
Miren Josu OmaetxebarriaAsier Fullaondo
Jesus Mari ArizmendiKerman Aloria
Eva Rodríguez SuárezFelix Elortza