title: Evaluating kinase ATP uptake and tyrosine phosphorylation using multiplexed quantification of chemically labeled and post-translationally modified peptides.
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.
authors.affiliation: Proteomics, Moffitt Cancer Center & Research Institute, 12902 Magnolia Drive, Tampa, FL 33612, United States.
authors.forename: Bin
authors.initials: B
authors.lastname: Fang
authors.affiliation: Molecular Oncology, Moffitt Cancer Center & Research Institute, 12902 Magnolia Drive, Tampa, FL 33612, United States.
authors.forename: Melissa A
authors.initials: MA
authors.lastname: Hoffman
authors.affiliation: Molecular Oncology, Moffitt Cancer Center & Research Institute, 12902 Magnolia Drive, Tampa, FL 33612, United States.
authors.forename: Abu-Sayeef
authors.initials: AS
authors.lastname: Mirza
authors.affiliation: Molecular Oncology, Moffitt Cancer Center & Research Institute, 12902 Magnolia Drive, Tampa, FL 33612, United States.
authors.forename: Katie M
authors.initials: KM
authors.lastname: Mishall
authors.affiliation: Chemical Biology and Molecular Medicine, Moffitt Cancer Center & Research Institute, 12902 Magnolia Drive, Tampa, FL 33612, United States.
authors.forename: Jiannong
authors.initials: J
authors.lastname: Li
authors.affiliation: Biomarker Research Initiative in Mass Spectrometry, ThermoFisher Scientific, Cambridge, MA, United States.
authors.forename: Scott M
authors.initials: SM
authors.lastname: Peterman
authors.affiliation: Cancer Biology and Evolution, Moffitt Cancer Center & Research Institute, 12902 Magnolia Drive, Tampa, FL 33612, United States.
authors.forename: Keiran S M
authors.initials: KS
authors.lastname: Smalley
authors.affiliation: Chemical Biology and Molecular Medicine, Moffitt Cancer Center & Research Institute, 12902 Magnolia Drive, Tampa, FL 33612, United States.
authors.forename: Kenneth H
authors.initials: KH
authors.lastname: Shain
authors.affiliation: Department of Otolaryngology, Center for Biotechnology and Genomic Medicine, Georgia Regents Cancer Center, Georgia Regents University, United States.
authors.forename: Paul M
authors.initials: PM
authors.lastname: Weinberger
authors.affiliation: Chemical Biology and Molecular Medicine, Moffitt Cancer Center & Research Institute, 12902 Magnolia Drive, Tampa, FL 33612, United States.
authors.forename: Jie
authors.initials: J
authors.lastname: Wu
authors.affiliation: Chemical Biology and Molecular Medicine, Moffitt Cancer Center & Research Institute, 12902 Magnolia Drive, Tampa, FL 33612, United States.
authors.forename: Uwe
authors.initials: U
authors.lastname: Rix
authors.affiliation: Chemical Biology and Molecular Medicine, Moffitt Cancer Center & Research Institute, 12902 Magnolia Drive, Tampa, FL 33612, United States.
authors.forename: Eric B
authors.initials: EB
authors.lastname: Haura
authors.affiliation: 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.
authors.forename: John M
authors.initials: JM
authors.lastname: Koomen
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D015415:Biomarkers / Q000032:analysis; D002853:Chromatography, Liquid / Q000379:methods*; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D010455:Peptides / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000032:analysis / Q000378:metabolism; D040901:Proteomics / Q000379:methods; D014443:Tyrosine / Q000378:metabolism*
chemical_list: D015415:Biomarkers; D010455:Peptides; D011506:Proteins; D014443:Tyrosine; D000255:Adenosine Triphosphate; D011494:Protein Kinases
keywords: Activity-based protein profiling; Cancer; Kinase signaling; Liquid chromatography–multiple reaction monitoring mass spectrometry (LC–MRM); Phosphotyrosine profiling; Spectral library