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

Methods. 2015 Jun 15;81:41-9. doi: 10.1016/j.ymeth.2015.03.006. Epub 2015 Mar 14.

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.

Keywords: Activity-based protein profiling; Cancer; Kinase signaling; Liquid chromatography–multiple reaction monitoring mass spectrometry (LC–MRM); Phosphotyrosine profiling; Spectral library.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Adenosine Triphosphate / metabolism*
  • Biomarkers / analysis
  • Chromatography, Liquid / methods*
  • Humans
  • Mass Spectrometry / methods*
  • Peptides / metabolism
  • Phosphorylation
  • Protein Kinases / metabolism*
  • Protein Processing, Post-Translational*
  • Proteins / analysis
  • Proteins / metabolism
  • Proteomics / methods
  • Tyrosine / metabolism*

Substances

  • Biomarkers
  • Peptides
  • Proteins
  • Tyrosine
  • Adenosine Triphosphate
  • Protein Kinases