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

g

g

g

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 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