A Strategy to Combine Sample Multiplexing with Targeted Proteomics Assays for High-Throughput Protein Signature Characterization

Brian K. Erickson, Christopher M. Rose, Craig R. Braun, Alison R. Erickson, Jeffrey Knott, Graeme C. McAlister, Martin Wühr, Joao A. Paulo, Robert A. Everley, Steven P. Gygi

Research output: Contribution to journalArticle

52 Scopus citations

Abstract

Targeted mass spectrometry assays for protein quantitation monitor peptide surrogates, which are easily multiplexed to target many peptides in a single assay. However, these assays have generally not taken advantage of sample multiplexing, which allows up to ten analyses to occur in parallel. We present a two-dimensional multiplexing workflow that utilizes synthetic peptides for each protein to prompt the simultaneous quantification of >100 peptides from up to ten mixed sample conditions. We demonstrate that targeted analysis of unfractionated lysates (2 hr) accurately reproduces the quantification of fractionated lysates (72 hr analysis) while obviating the need for peptide detection prior to quantification. We targeted 131 peptides corresponding to 69 proteins across all 60 National Cancer Institute cell lines in biological triplicate, analyzing 180 samples in only 48 hr (the equivalent of 16 min/sample). These data further elucidated a correlation between the expression of key proteins and their cellular response to drug treatment.

Original languageEnglish (US)
Pages (from-to)361-370
Number of pages10
JournalMolecular Cell
Volume65
Issue number2
DOIs
StatePublished - Jan 19 2017

All Science Journal Classification (ASJC) codes

  • Molecular Biology
  • Cell Biology

Keywords

  • NCI-60
  • cancer
  • high-throughput
  • isobaric tag
  • multiplexing
  • proteomics
  • targeted proteomics

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    Erickson, B. K., Rose, C. M., Braun, C. R., Erickson, A. R., Knott, J., McAlister, G. C., Wühr, M., Paulo, J. A., Everley, R. A., & Gygi, S. P. (2017). A Strategy to Combine Sample Multiplexing with Targeted Proteomics Assays for High-Throughput Protein Signature Characterization. Molecular Cell, 65(2), 361-370. https://doi.org/10.1016/j.molcel.2016.12.005