RT Journal Article
SR Electronic
T1 Motif-centric phosphoproteomics to target kinase-mediated signaling pathways
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2021.07.02.450911
DO 10.1101/2021.07.02.450911
A1 Chia-Feng Tsai
A1 Kosuke Ogata
A1 Naoyuki Sugiyama
A1 Yasushi Ishihama
YR 2021
UL http://biorxiv.org/content/early/2021/07/04/2021.07.02.450911.abstract
AB 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 reaction is used to generate the 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. Over 7,000 tyrosine phosphorylation sites were quantified from several tens of µg of starting materials. This approach enables the quantification of multiple phosphorylation pathways under physiological or pathological regulation in a motif-centric manner.Motivation Sensitivity for detecting phosphopeptides with a particular phosphorylation motif is limited, especially for tyrosine phosphopeptides.Competing Interest StatementThe authors have declared no competing interest.