Dataset Information

Motif-centric phosphoproteomics to target kinase-mediated signaling pathways

ABSTRACT: 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.

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Yasushi Ishihama

PROVIDER: PXD026996 | JPOST Repository | Wed Dec 01 00:00:00 GMT 2021

REPOSITORIES: jPOST

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Dataset's files

Source:

			Action	DRS
	170620Fe_3plex_PV100_MS2.raw	Raw
	170620Fe_3plex_PV150_MS2.raw	Raw
	170620Fe_3plex_PV50_MS2.raw	Raw
	170620Fe_3plex_noPV_MS2.raw	Raw
	170705Fe_DCKi_CK2_TMT6_1_MS3.raw	Raw

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Publications

Motif-centric phosphoproteomics to target kinase-mediated signaling pathways.

Tsai Chia-Feng CF Ogata Kosuke K Sugiyama Naoyuki N Ishihama Yasushi Y

Cell reports methods 20220114 1

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, ...[more]

PMID: 35474870

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Project description:Phosphorylation is a widespread post-translational modification that regulates numerous biological processes. Viruses can alter the physiological activities of host cells to promote virus particle replication, and manipulating phosphorylation is one of the mechanisms. Senecavirus A (SVA) the causative agent of porcine idiopathic vesicular disease. Although numerous studies on SVA have been performed, comprehensive phosphoproteomics analysis of SVA infection is lacking. In the present study, we performed a quantitative mass spectrometry-based phosphoproteomics survey of SVA infection in instituto biologico-rim suino-2 (IBRS-2) cells. Three parallel experiments were performed and 4520 phosphosites were identified on 2084 proteins. Gene Ontology functional enrichment and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses showed that many of the phosphorylated proteins were involved in apoptosis and spliceosome pathways, and subcellular structure localisation analysis revealed that more than half were located in the nucleus. Motif analysis of proteins with differentially regulated phosphosites showed that proline, aspartic acid and glutamic acid were the most abundant residues in the serine motif, while proline and arginine were the most abundant in the threonine motif. Forty phosphosites on 27 proteins were validated by parallel reaction monitoring phosphoproteomics, and 30 phosphosites in 21 proteins were verified. Nine proteins with significantly altered phosphosites were further explored, and eight (SRRM2, CDK13, DDX20, DDX21, BAD, ELAVL1, PBK, and STAT3) may play a role in SVA infection. Finally, identified eight predicted kinases, and the kinase activity of inhibitor of nuclear factor kappa-B kinase subunit β and CDK9 was reversed following SVA infection. This is the first phosphoproteomics analysis of SVA infection of IBRS-2 cells, and the results greatly expand our knowledge of SVA infection. The findings provide a basis for studying the interactions of other picornaviruses and their mammalian host cells.