title: An Optimized Chromatographic Strategy for Multiplexing In Parallel Reaction Monitoring Mass Spectrometry: Insights from Quantitation of Activated Kinases.
abstract: Reliable quantitation of protein abundances in defined sets of cellular proteins is critical to numerous biological applications. Traditional immunodetection-based methods are limited by the quality and availability of specific antibodies, especially for site-specific post-translational modifications. Targeted proteomic methods, including the recently developed parallel reaction monitoring (PRM) mass spectrometry, have enabled accurate quantitative measurements of up to a few hundred specific target peptides. However, the degree of practical multiplexing in label-free PRM workflows remains a significant limitation for the technique. Here we present a strategy for significantly increasing multiplexing in label-free PRM that takes advantage of the superior separation characteristics and retention time stability of meter-scale monolithic silica-C18 column-based chromatography. We show the utility of the approach in quantifying kinase abundances downstream of previously developed active kinase enrichment methodology based on multidrug inhibitor beads. We examine kinase activation dynamics in response to three different MAP kinase inhibitors in colorectal carcinoma cells and demonstrate reliable quantitation of over 800 target peptides from over 150 kinases in a single label-free PRM run. The kinase activity profiles obtained from these analyses reveal compensatory activation of TGF-β family receptors as a response to MAPK blockade. The gains achieved using this label-free PRM multiplexing strategy will benefit a wide array of biological applications.
authors.affiliation: From the ‡Department of Pathology, University of California San Francisco, San Francisco, California; anatoly.urisman@ucsf.edu.|§Department of Pharmaceutical Chemistry, University of California San Francisco, San Francisco, California.
authors.forename: Anatoly
authors.identifier: 0000-0001-8364-5303
authors.initials: A
authors.lastname: Urisman
authors.affiliation: §Department of Pharmaceutical Chemistry, University of California San Francisco, San Francisco, California.|¶Department of Cellular and Molecular Pharmacology, University of California San Francisco, San Francisco, California.
authors.forename: Rebecca S
authors.initials: RS
authors.lastname: Levin
authors.affiliation: ¶Department of Cellular and Molecular Pharmacology, University of California San Francisco, San Francisco, California.|‖Department of Medicine, University of California San Francisco, San Francisco, California.
authors.forename: John D
authors.initials: JD
authors.lastname: Gordan
authors.affiliation: **Department of Bioengineering and Therapeutic Sciences, University of California San Francisco, San Francisco, California.
authors.forename: James T
authors.initials: JT
authors.lastname: Webber
authors.affiliation: §Department of Pharmaceutical Chemistry, University of California San Francisco, San Francisco, California.
authors.forename: Hilda
authors.initials: H
authors.lastname: Hernandez
authors.affiliation: ‡‡Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan.
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Ishihama
authors.affiliation: ¶Department of Cellular and Molecular Pharmacology, University of California San Francisco, San Francisco, California.|§§Howard Hughes Medical Institute, University of California San Francisco, San Francisco, California.
authors.forename: Kevan M
authors.initials: KM
authors.lastname: Shokat
authors.affiliation: §Department of Pharmaceutical Chemistry, University of California San Francisco, San Francisco, California.
authors.forename: Alma L
authors.initials: AL
authors.lastname: Burlingame
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D002853:Chromatography, Liquid / Q000379:methods; D015179:Colorectal Neoplasms / Q000201:enzymology*; D004789:Enzyme Activation; D045325:HCT116 Cells; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D051379:Mice; D010455:Peptides / Q000032:analysis; D010770:Phosphotransferases / Q000032:analysis*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D040901:Proteomics / Q000379:methods*; D057188:Workflow
chemical_list: D010455:Peptides; D047428:Protein Kinase Inhibitors; D010770:Phosphotransferases