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

Anatoly Urisman,Rebecca S Levin,John D Gordan,James T Webber,Hilda Hernandez,Yasushi Ishihama,Kevan M Shokat,Alma L Burlingame

doi:10.1074/mcp.m116.058172

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.

Highlights

From the ‡Department of Pathology, University of California San Francisco, San Francisco, California; §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; ʈDepartment of Medicine, University of California San Francisco, San Francisco, California; **Department of Bioengineering and Therapeutic Sciences, University of California San Francisco, San Francisco, California; ‡‡Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan; §§Howard Hughes Medical Institute, University of California San Francisco, San Francisco, California
Monolithic Silica-C18 Column Performance in 6-h dependent acquisition (DDA) Gradients—As the first step toward developing a highly multiplexed parallel reaction monitoring (PRM) pipeline, we examined the performance of a 200 cm monolithic silica-C18 column in a variety of conditions by considering three factors: estimating its peak capacity (PC), peak width, retention time (RT) reproducibility, and sensitivity of detection (Fig. 2)
We have developed a strategy for highly multiplexed PRM relying on ultra-long monolithic silica-C18 column based liquid chromatography and demonstrated its utility by quantifying the activity of a broad segment of the human kinome in vivo in response to kinase inhibitor therapy

Summary

Introduction

From the ‡Department of Pathology, University of California San Francisco, San Francisco, California; §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; ʈDepartment of Medicine, University of California San Francisco, San Francisco, California; **Department of Bioengineering and Therapeutic Sciences, University of California San Francisco, San Francisco, California; ‡‡Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan; §§Howard Hughes Medical Institute, University of California San Francisco, San Francisco, California. We and other groups have previously reported on the development and use of Multidrug Inhibitor Beads (MIB) strategy for active kinase enrichment (4 – 8) In this approach, kinase affinity enrichment scaffolds based on known kinase inhibitor structures targeted to conformationally active kinases are chemically coupled to a resin, which is used to capture broad classes of kinases from cell lysates (see recent review of this and related approaches by Daub [4]). Chromatographic Strategy for Multiplexing in PRM coupled to quantitative mass spectrometry-based proteomics, MIB enrichment can be used to determine kinase inhibitor specificity by comparing relative abundances of particular kinases enriched from drug treated and untreated samples or to study kinase activation dynamics as response to clinical therapy or various biological perturbations. MIB experiments have demonstrated on-target suppression of kinase activity and activation of compensatory kinase signaling pathways driving resistance to therapy in triple negative breast cancer [5], drug-resistant leukemia [6], and ERBB2positive breast cancer [7]

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Conclusion