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

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

Author information

1
Proteomics, Moffitt Cancer Center & Research Institute, 12902 Magnolia Drive, Tampa, FL 33612, United States.
2
Molecular Oncology, Moffitt Cancer Center & Research Institute, 12902 Magnolia Drive, Tampa, FL 33612, United States.
3
Chemical Biology and Molecular Medicine, Moffitt Cancer Center & Research Institute, 12902 Magnolia Drive, Tampa, FL 33612, United States.
4
Biomarker Research Initiative in Mass Spectrometry, ThermoFisher Scientific, Cambridge, MA, United States.
5
Cancer Biology and Evolution, Moffitt Cancer Center & Research Institute, 12902 Magnolia Drive, Tampa, FL 33612, United States.
6
Department of Otolaryngology, Center for Biotechnology and Genomic Medicine, Georgia Regents Cancer Center, Georgia Regents University, United States.
7
Molecular Oncology, Moffitt Cancer Center & Research Institute, 12902 Magnolia Drive, Tampa, FL 33612, United States; Chemical Biology and Molecular Medicine, Moffitt Cancer Center & Research Institute, 12902 Magnolia Drive, Tampa, FL 33612, United States. Electronic address: john.koomen@moffitt.org.

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

PMID:
25782629
PMCID:
PMC4460980
DOI:
10.1016/j.ymeth.2015.03.006
[Indexed for MEDLINE]
Free PMC Article

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