Evaluating kinase ATP uptake and tyrosine phosphorylation using multiplexed quantification of chemically labeled and post-translationally modified peptides

Bin Fang, Melissa A. Hoffman, Abu Sayeef Mirza, Katie M. Mishall, Jiannong Li, Scott M. Peterman, Keiran S.M. Smalley, Kenneth H. Shain, Paul M. Weinberger, Jie Wu, Uwe Rix, Eric B. Haura, John M. Koomen

Research output: Contribution to journalArticle

10 Scopus citations

Abstract

Cancer biologists and other healthcare researchers face an increasing challenge in addressing the molecular complexity of disease. Biomarker measurement tools and techniques now contribute to both basic science and translational research. In particular, liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM) for multiplexed measurements of protein biomarkers has emerged as a versatile tool for systems biology. Assays can be developed for specific peptides that report on protein expression, mutation, or post-translational modification; discovery proteomics data rapidly translated into multiplexed quantitative approaches. Complementary advances in affinity purification enrich classes of enzymes or peptides representing post-translationally modified or chemically labeled substrates. Here, we illustrate the process for the relative quantification of hundreds of peptides in a single LC-MRM experiment. Desthiobiotinylated peptides produced by activity-based protein profiling (ABPP) using ATP probes and tyrosine-phosphorylated peptides are used as examples. These targeted quantification panels can be applied to further understand the biology of human disease.

Original languageEnglish (US)
Pages (from-to)41-49
Number of pages9
JournalMethods
Volume81
DOIs
StatePublished - Jun 15 2015

Keywords

  • Activity-based protein profiling
  • Cancer
  • Kinase signaling
  • Liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM)
  • Phosphotyrosine profiling
  • Spectral library

ASJC Scopus subject areas

  • Molecular Biology
  • Biochemistry, Genetics and Molecular Biology(all)

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    Fang, B., Hoffman, M. A., Mirza, A. S., Mishall, K. M., Li, J., Peterman, S. M., Smalley, K. S. M., Shain, K. H., Weinberger, P. M., Wu, J., Rix, U., Haura, E. B., & Koomen, J. M. (2015). Evaluating kinase ATP uptake and tyrosine phosphorylation using multiplexed quantification of chemically labeled and post-translationally modified peptides. Methods, 81, 41-49. https://doi.org/10.1016/j.ymeth.2015.03.006