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

ACCESS DATA

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:Protein sulfation can be crucial in regulating protein-protein interactions but remains largely underexplored. Sulfation is near-isobaric to phosphorylation, making it particularly challenging to investigate using mass spectrometry. The degree to which tyrosine sulfation (sY) is misidentified as phosphorylation (pY) is thus an unresolved concern. This study explores the extent of sY misidentification within the human phosphoproteome by distinguishing between sulfation and phosphorylation based on their mass difference. Using Gaussian mixture models (GMMs), we screened ~45M peptide-spectrum matches (PSMs) from the PeptideAtlas Human Phosphoproteome build for peptidoforms with mass error shifts indicative of sulfation. This analysis pinpointed 104 candidate sulfated peptidoforms, backed-up by Gene Ontology (GO) terms and custom terms linked to sulfation. False positive filtering by manual annotation resulted in 31 convincing peptidoforms spanning 7 known and 7 novel sY sites. Y47 in Calumenin was particularly intriguing since mass error shifts, acidic motif conservation, and MS2 neutral loss patterns characteristic of sulfation, but not phosphorylation, provided strong evidence that this site can only be sulfated. Overall, although misidentification of sulfation in phosphoproteomics datasets derived from cell and tissue intracellular extracts can occur, it appears relatively rare and should not be considered a confounding factor for high-quality phosphoproteomics studies. Protein sulfation can be crucial in regulating protein-protein interactions but remains largely underexplored. Sulfation is near-isobaric to phosphorylation, making it particularly challenging to investigate using mass spectrometry. The degree to which tyrosine sulfation (sY) is misidentified as phosphorylation (pY) is thus an unresolved concern. This study explores the extent of sY misidentification within the human phosphoproteome by distinguishing between sulfation and phosphorylation based on their mass difference. Using Gaussian mixture models (GMMs), we screened ~45M peptide-spectrum matches (PSMs) from the PeptideAtlas Human Phosphoproteome build for peptidoforms with mass error shifts indicative of sulfation. This analysis pinpointed 104 candidate sulfated peptidoforms, backed-up by Gene Ontology (GO) terms and custom terms linked to sulfation. False positive filtering by manual annotation resulted in 31 convincing peptidoforms spanning 7 known and 7 novel sY sites. Y47 in Calumenin was particularly intriguing since mass error shifts, acidic motif conservation, and MS2 neutral loss patterns characteristic of sulfation, but not phosphorylation, provided strong evidence that this site can only be sulfated. Overall, although misidentification of sulfation in phosphoproteomics datasets derived from cell and tissue intracellular extracts can occur, it appears relatively rare and should not be considered a confounding factor for high-quality phosphoproteomics studies.