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 Maurice Weinberger, Jie Wu, Uwe Rix, Eric B. Haura, John M. Koomen

Research output: Contribution to journalArticle

9 Citations (Scopus)

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 - Jan 1 2015

Fingerprint

Phosphorylation
Tyrosine
Phosphotransferases
Adenosine Triphosphate
Peptides
Liquid chromatography
Biomarkers
Liquid Chromatography
Mass spectrometry
Mass Spectrometry
Proteins
Translational Medical Research
Systems Biology
Monitoring
Post Translational Protein Processing
Proteomics
Purification
Assays
Research Personnel
Delivery of Health Care

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)

Cite this

Evaluating kinase ATP uptake and tyrosine phosphorylation using multiplexed quantification of chemically labeled and post-translationally modified peptides. / Fang, Bin; Hoffman, Melissa A.; Mirza, Abu Sayeef; Mishall, Katie M.; Li, Jiannong; Peterman, Scott M.; Smalley, Keiran S.M.; Shain, Kenneth H.; Weinberger, Paul Maurice; Wu, Jie; Rix, Uwe; Haura, Eric B.; Koomen, John M.

In: Methods, Vol. 81, 01.01.2015, p. 41-49.

Research output: Contribution to journalArticle

Fang, B, Hoffman, MA, Mirza, AS, Mishall, KM, Li, J, Peterman, SM, Smalley, KSM, Shain, KH, Weinberger, PM, Wu, J, Rix, U, Haura, EB & Koomen, JM 2015, 'Evaluating kinase ATP uptake and tyrosine phosphorylation using multiplexed quantification of chemically labeled and post-translationally modified peptides', Methods, vol. 81, pp. 41-49. https://doi.org/10.1016/j.ymeth.2015.03.006
Fang, Bin ; Hoffman, Melissa A. ; Mirza, Abu Sayeef ; Mishall, Katie M. ; Li, Jiannong ; Peterman, Scott M. ; Smalley, Keiran S.M. ; Shain, Kenneth H. ; Weinberger, Paul Maurice ; Wu, Jie ; Rix, Uwe ; Haura, Eric B. ; Koomen, John M. / Evaluating kinase ATP uptake and tyrosine phosphorylation using multiplexed quantification of chemically labeled and post-translationally modified peptides. In: Methods. 2015 ; Vol. 81. pp. 41-49.
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