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Mol Cell Proteomics. 2017 Feb;16(2):265-277. doi: 10.1074/mcp.M116.058172. Epub 2016 Dec 11.

An Optimized Chromatographic Strategy for Multiplexing In Parallel Reaction Monitoring Mass Spectrometry: Insights from Quantitation of Activated Kinases.

Author information

1
From the ‡Department of Pathology, University of California San Francisco, San Francisco, California; anatoly.urisman@ucsf.edu.
2
§Department of Pharmaceutical Chemistry, University of California San Francisco, San Francisco, California.
3
¶Department of Cellular and Molecular Pharmacology, University of California San Francisco, San Francisco, California.
4
‖Department of Medicine, University of California San Francisco, San Francisco, California.
5
**Department of Bioengineering and Therapeutic Sciences, University of California San Francisco, San Francisco, California.
6
‡‡Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan.
7
§§Howard Hughes Medical Institute, University of California San Francisco, San Francisco, California.

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.

PMID:
27940637
PMCID:
PMC5294213
DOI:
10.1074/mcp.M116.058172
[Indexed for MEDLINE]
Free PMC Article

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