Pme5 - Csic

Pme5 - Csic

Proteored Multilab Experiment 5 QUANTITATIVE PROTEOMICS 4 proteins spiked in E. coli protein matrix Progenesis LC-MS assisted label-free analysis 16 Marzo 2010 WG1&WG2 Meeting - CIC Salamanca 1 Labs Reporting Data Universidad de Alicante (Qual-Quant) Hospital Universitario Instituto de Oncologia Vall dHebron (Qual-Quant) Centro de Biologa Molecular Severo Ochoa (Qual: 72 Ecoli + 2 spiked) 16 Marzo 2010 WG1&WG2 Meeting - CIC Salamanca 2 APPROACH LAB1-LAB2 Sample processing and Data acquisition Liquid digestion (iodoacetamide, trypsin) nLC-nESI-IT 5 - 4 runs of sample A, 4 runs of sample B Label-free analysis with Progenesis LC-MS LC-MS plot alignment Whole-feature selection feature abundance >1E+06 Mascot search (2mclv, Ox(M), Deam(QN), 1.2Da 1.4 Da parent, 0.6Da 0.7 Da fragment) Peptide match validation: score > 25, matching E. coli or spiked protein 16 Marzo 2010 WG1&WG2 Meeting - CIC Salamanca 3 Reference LC-MS run selection 16 Marzo 2010

WG1&WG2 Meeting - CIC Salamanca 4 LC-MS runs alignment 16 Marzo 2010 WG1&WG2 Meeting - CIC Salamanca 5 Feature filtering 16 Marzo 2010 WG1&WG2 Meeting - CIC Salamanca 6 Stats-based feature selection 16 Marzo 2010 WG1&WG2 Meeting - CIC Salamanca 7 Selected feature stats 16 Marzo 2010 WG1&WG2 Meeting - CIC Salamanca 8 Peptide search: mgf export, xml import 16 Marzo 2010 WG1&WG2 Meeting - CIC Salamanca 9

Peptide filtering 16 Marzo 2010 WG1&WG2 Meeting - CIC Salamanca 10 Protein view - conflict resolution 16 Marzo 2010 WG1&WG2 Meeting - CIC Salamanca 11 Report configuration 16 Marzo 2010 WG1&WG2 Meeting - CIC Salamanca 12 Web report 16 Marzo 2010 WG1&WG2 Meeting - CIC Salamanca 13 Average and %CV of A/B ratios of groups of E. coli proteins identified-quantified with only avg ratio 1.20 1.20 0.80 0.60 0.40 1.00 0.80

average A/B average A/B 0.60 0.40 0.20 0.20 0.00 1 peptide 2 peptides 0.80 lab2 0.60 lab1 0.40 0.20 0.00 1&2 3-7 peptides2 peptides >7 peptides 1&2 1 peptide peptides peptides %cv 0.00 2 peptides 3-7 1 peptide >7 peptides peptides 80 80 70 70 70

60 60 60 average A/B 80 50 40 30 20 10 50 40 30 average A/B average A/B 1.20 1.00 1.00 %CV of A/B avg ratio 20 1 peptide 2 peptides 16 Marzo 2010 3-7 peptides >7 peptides 50 40 lab2 30

lab1 20 10 10 0 1&2 peptides %cv 0 1&2 peptides 3-7 peptides >7 peptides 1 peptide 2 peptides 1&2 peptides 0 1 peptide 2 peptides 3-7 >7 peptides peptides WG1&WG2 Meeting - CIC Salamanca 1&2 peptides 3-7 peptides >7 peptides 14 Deviation of average abundance LAB1 ALDOA MYG CYC 25fm 200fm 1000fm 160 140 120 PME5A PME5B

spiked A 100 spiked B 80 60 40 20 0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 P EP TIDES USED FOR ID-QUANT 16 Marzo 2010 WG1&WG2 Meeting - CIC

Salamanca 15 Deviation of average abundance LAB2 ALDOA BSA/MYG CYC 25fm 1fm/200fm 1000fm 70 60 50 PME5A PME5B 40 %CV SPIKED A SPIKED B 30 20 10 0 0 2 4 6 8 10

12 PEPTIDES USED FOR ID-QUANT 16 Marzo 2010 WG1&WG2 Meeting - CIC Salamanca 16 PROTEINS IDENTIFIED WITH AT LEAST 1 PEPTIDE LAB2 LAB1 17 35 30 50 25 only LAB1 only LAB2 20 LAB1 & LAB2 15 10 5 78 0 16 Marzo 2010 P ROTEIN WG1&WG2 MeetingN- CIC Salamanca 17 PROTEINS IDENTIFIED WITH AT LEAST 2 PEPTIDES IN ONE LAB

LAB2 LAB1 9 35 30 only LAB1 25 17 20 only LAB2 LAB1 & LAB2 15 10 46 5 0 16 Marzo 2010 E I N N - CIC WG1&WG2PROT Meeting Salamanca 18 Proteins stats LAB1 ALDOA MYG CYC %CV OF AVERAGE RATIO 25fm 200fm 1000fm

50 40 67 Number of E Coli single hit- proteins id 14 Number of Spiked proteins id. 3 30 Peptide count Confidence score Anova (p)* CYC_HORSE 7 287.88 0.0053064 0.6 0.67 10 MYG_EQUBU 4 236.17 0.00068157 5.5 2.6 0 ALDOA_RABIT 3

152.12 0.00028094 3.8 2.0 BSA_BOVIN K2C1_HUMAN 4 NOT 192.23 FOUND 0.26933846 1.4 4 6 Accession 20 0 1 2 3 4 5 6 7 8 9 10 UNIQUE PEPTIDES USED AT LEAST AVERAGE RATIO (log 2)

80 #PROTEINS ID-QUANT Number of E Coli proteins id. (total) 70 60 50 40 30 20 10 0 016 Marzo 1 22010 3 1 0.8 0.6 0.4 0.2 0 -0.2 -0.4 -0.6 -0.8 -1 WG1&WG2 8 9 10 Meeting0- CIC Salamanca UNIQUE PEPTIDES USED AT LEAST 4 5 6 7 A/B dtn - prep

2 UNIQUE PEPTIDES USED AT LEAST 8 19 10 Proteins stats LAB2 ALDOA BSA/MYG CYC %CV of average A/B 25fm 1fm/200fm 1000fm Number of E Coli proteins id. (total) 128 70 Number of E Coli single hit- proteins id 74 60 Number of Spiked proteins id. 4 50 40 Peptide count Confidence score Anova (p)* A/B dtn - prep CYC_HORSE

5 285.88 0.0000067 0.57 0.67 10 MYG_EQUBU 3 244.41 0.00010617 2.18 2.6 0 ALDOA_RABIT 1 34.81 0.9008034 0.99 2.0 BSA_BOVIN 3 174.11 0.0105259 1.22 0.2 30 Accession 20 0

2 4 6 UNIQUE PEPTIDES USED AT LEAST 8 10 120 AVERAGE A/B (log2) #PROTEINS ID-QUANT 140 100 80 60 40 20 0 16 0 Marzo 2010 2 1 0.8 0.6 0.4 0.2 0 -0.2 -0.4 -0.6 -0.8 -1 WG1&WG2 8 10 Meeting -0 CIC Salamanca UNIQUE PEPTIDES USED AT LEAST 4 6

2 4 6 UNIQUE PEPTIDES USED AT LEAST 8 20 10 AVERAGE A/B (log2) 1 0.8 0.6 0.4 0.2 0 -0.2 -0.4 -0.6 -0.8 -1 lab2 lab1 0 4 6 8 10 UNIQUE PEPTIDES USED AT LEAST 70 %CV of average A/B 2 60 50

40 lab2 30 lab1 20 10 0 0 4 6 8 10 UNIQUE PEPTIDES USED AT LEAST 140 #PROTEINS ID-QUANT 2 120 100 80 lab2 60 lab1 40 20 0 16 Marzo 2010 0 2 4 6

8 WG1&WG2 Meeting CIC UNIQUE PEPTIDES USED AT -LEAST Salamanca 10 21 LINEAR MODEL PREDICTION OF PEPTIDE COUNTS 8 y = 0.004x + 3.0359 R2 = 0.9946 7 PEPTIDE COUNTS CYC_HORSE 1000 1200 7 MYG_EQUBU 200 340 4 ALDOA_RABIT 25 98 3

1 6.6 2-3 BSA_BOVIN y = 0.0036x + 2.7052 R2 = 0.999 4 concentration /10 (pg/microg) Accession 6 5 concentration (fmol/microg) Peptide count 3 2 Suitable for interpolation 1 0 0 200 400 600 800 1000

1200 1400 PROTEIN CONCENTRATION concentration (fmol/microg) 16 Marzo 2010 concentration/10 (pg/microg) WG1&WG2 Meeting - CIC Salamanca 22 PREDICTION OF PEPTIDE COUNTS LOGARITHMIC MODEL 8 7 y = 1.0556Ln(x) - 0.7609 R2 = 0.8795 6 concentration /10 (pg/microg) CYC_HORSE 1000 1200 7 MYG_EQUBU 200 340 4 ALDOA_RABIT 25

98 3 1 6.6 0 Accession 5 PEPTIDE COUNTS concentration (fmol/microg) 4 BSA_BOVIN 3 Peptide count y = 1.5985Ln(x) - 4.6602 R2 = 0.9252 2 1 0 0.1 1 10 100 1000 10000 -1 Suitable for extrapolation? -2

-3 PROTEIN CONCENTRATION concentration (fmol/microg) 16 Marzo 2010 concentration/10 (pg/microg) WG1&WG2 Meeting - CIC Salamanca 23 PREDICTION OF PEPTIDE COUNTS EXPONENTIAL MODEL 8 7 PEPTIDE COUNTS concentration /10 (pg/microg) CYC_HORSE 1000 1200 7 MYG_EQUBU 200 340 4 ALDOA_RABIT 25 98 3

1 6.6 1 Accession 0.2253 y = 1.3748x R2 = 0.9356 6 concentration (fmol/microg) 5 BSA_BOVIN 4 Peptide count y = 0.6078x0.3385 R2 = 0.9682 3 2 Suitable for inter- and extrapolation? 1 0 0.1 1 10 100 1000

10000 PROTEIN CONCENTRATION concentration (fmol/microg) 16 Marzo 2010 concentration/10 (pg/microg) WG1&WG2 Meeting - CIC Salamanca 24 CONCLUSIONS LC-MS label free method was successfully used to quantitate relative abundance of spiked proteins across samples in a matrix of medium complexity Low resolution instruments (ESI-IT) produced reasonably accurate results Some spiked proteins can be in or below the LOD of the used instruments (inclusion lists) A thresold of 3 unique peptides seem to be needed for achieving an acceptable dispersion (p.ex. CV< 20%) of protein abundance data used for quantitation 16 Marzo 2010 WG1&WG2 Meeting - CIC Salamanca 25

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