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ABRF sPRG 2013 contest: Summary of ProteoRed results

ABRF sPRG 2013 contest: Summary of ProteoRed results. CIMA, University of Navarra, Pamplona-Iruña, 10th of December 2013. General considerations.

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ABRF sPRG 2013 contest: Summary of ProteoRed results

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  1. ABRF sPRG 2013 contest: Summary of ProteoRed results CIMA, University of Navarra, Pamplona-Iruña, 10th of December 2013

  2. General considerations • sPRG is developing a new proteomic standard, derived from a series of stable isotope-labeled (“heavy”) peptides as internal standards conserved across Homo sapiens, Musmusculus and Rattusnorvegicus. • collaborative study that focuses on the ability of core facilities to determine relative quantitation of up to 1,000 heavy/light peptide pairs in a single sample.

  3. Sample characteristics • Sequences of synthetic peptides were derived from approximately 552 proteins, conserved across proteomes of commonly analyzed species: Homo sapiens, Musmusculus and Rattusnorvegicus. • single vial containing a lyophilized mixture of stable isotope labeled (SIL) synthetic peptides combined with a tryptic digest of HEK293 cells. • sufficient quantities (5 μg digested lysate and 375 fmol of each heavy peptide) to permit at least three technical replicates.

  4. primary goals of this study • To allow labs to evaluate their ability to measure heavy/light peptide ratios for a large number of peptides in a complex biological background. • To provide a learning opportunity for labs unfamiliar with experimental techniques and data analysis strategies for measuring heavy/light peptide ratios. • To benchmark the standard across a large number of labs. • Participants are asked to analyze the sample in triplicate using the LC-MS instrument platform of their choice and report peak area ratios and coefficients of variation.

  5. Suggested methodology

  6. Peptidesidentified and quantifiedbyProteoRedparticipants: similar values as thosedescribedby ABRF testers

  7. Strongcorrelation of retention times suggestsgoodpeptideidentification performance

  8. HETEROGENEITY IN THE CALCULATION OF THE RATIO

  9. Differentdegrees of correlationbetweenpairs of data sets (1)

  10. Differentdegrees of correlationbetweenpairs of data sets (2) Pearson coefficient: +/- 0<p<+/-0.4 poor correlation +/- 0.4<p<+/-0.6 medium correlation +/- 0.6<p<+/-1 good correlation

  11. Proposalfor a new WG1/WG2 multicentricexperiment Hahne H, Pachl F, Ruprecht B, Maier SK, Klaeger S, Helm D, Médard G, Wilm M, Lemeer S, Kuster B. DMSO enhances electrospray response, boosting sensitivity of proteomic experiments. Nat Methods. 2013 Oct;10(10):989-91. doi: 10.1038/nmeth.2610. Epub 2013 Aug 25. PubMed PMID: 23975139.

  12. Bruker AmaZon ion trap; effect of 4% DMSO Sample: 150 fmol of 6 trypsin-digested proteins

  13. Bruker AmaZon ion trap; effect of 4% DMSO Sample: 1 mg of UPSII standard

  14. MRM ANALYSIS: 10 fmol of beta-gal digest in 5500 QTRAP (-DMSO)

  15. MRM ANALYSIS: 10 fmol of beta-gal digest in 5500 QTRAP (+DMSO)

  16. Proposal for a new PME experiment: Sample: PME8 (preferred) or PME9 Aim: using SRM/MRM (true or pseudo), compare areas corresponding to a set of previously selected peptide transitions both in absence or presence of DMSO (4%-5%). Objective: demonstrate significative (2-3 fold) increase in the signals as well as a significative increase in the quality of the quantitative data. Schedule: sample delivery: april 2014 Analysis: may-june 2014 Data: send in july 2014 Data Analysis: october-november 2014

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