Cell Reports Methods
Volume 2, Issue 1, 24 January 2022, 100138
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Article
Motif-centric phosphoproteomics to target kinase-mediated signaling pathways

https://doi.org/10.1016/j.crmeth.2021.100138Get rights and content
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Highlights

  • Successful generation of motif-centric carrier peptides by in vitro kinase reactions

  • Subsequent tandem mass tag quantification targeted to motif-centric phosphopeptides

  • Successful quantitation of more than 7,000 pY sites from 25 μg of peptides achieved

  • Multiple kinase-mediated pathways can be targeted for TMT quantitation

Motivation

Alterations in protein phosphorylation signaling networks associated with the dysregulation of kinase activity are closely linked to a variety of pathological conditions. However, quantitative and multiplexable assays to reveal the phosphorylation network based on kinase-substrate relationships do not exist for rare cell populations. To address this issue, we have developed an approach in which the signals of intrinsic kinase substrates are boosted using isobaric labeling that targets phosphopeptides generated in in vitro kinase reactions. As a result, we succeeded in encompassing specific kinase substrates in a motif-centric manner without the need for immunoprecipitation.

Summary

Identifying cellular phosphorylation pathways based on kinase-substrate relationships is a critical step to understanding the regulation of physiological functions in cells. Mass spectrometry-based phosphoproteomics workflows have made it possible to comprehensively collect information on individual phosphorylation sites in a variety of samples. However, there is still no generic approach to uncover phosphorylation networks based on kinase-substrate relationships in rare cell populations. Here, we describe a motif-centric phosphoproteomics approach combined with multiplexed isobaric labeling, in which in vitro kinase reactions are used to generate targeted phosphopeptides, which are spiked into one of the isobaric channels to increase detectability. Proof-of-concept experiments demonstrate selective and comprehensive quantification of targeted phosphopeptides by using multiple kinases for motif-centric channels. More than 7,000 tyrosine phosphorylation sites were quantified from several tens of micrograms of starting materials. This approach enables the quantification of multiple phosphorylation pathways under physiological or pathological regulation in a motif-centric manner.

Keywords

motif-centric
phosphoproteome
in vitro kinase reaction
phosphopeptide enrichment
isobaric tag
boosting MS signal
tyrosine phosphoproteome
TMT quantitation
EGFR signaling network
kinase-substrate relationship

Data and code availability

  • Data described in this paper have been deposited at https://zenodo.org and are publicly available as of the date of publication. DOIs are listed in the key resources table.

  • This paper does not report original code.

  • Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request

Cited by (0)

3

These authors contributed equally

4

Present address: Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA 99354, USA

5

Lead contact