4 research outputs found

    Multiplexed Phosphoproteomic Profiling Using Titanium Dioxide and Immunoaffinity Enrichments Reveals Complementary Phosphorylation Events

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    A comprehensive view of protein phosphorylation remains an unmet challenge in the field of cell biology. Mass spectrometry-based proteomics is one of the most promising approaches for identifying thousands of phosphorylation events in a single experiment, yet the full breadth of the phosphoproteome has yet to be elucidated. In this article, we examined the complementarity of two methods for phosphopeptide enrichment based on either titanium dioxide (TiO<sub>2</sub>) enrichment or phosphorylation motif-specific immunoaffinity precipitation (IAP) with four different antibodies. Each method identified nearly 2000 phosphoproteins. However, distinct populations of phosphopeptides were observed. Despite quantifying over 10 000 unique phosphorylation events using TiO<sub>2</sub> and over 3900 with IAP, less than 5% of the sites were in common. Agreeing with published literature, the ratio of pS:pT:pY phosphorylation for the TiO<sub>2</sub>-enriched data set approximated 90:10:<1. In contrast, that ratio for the combined IAP data sets was 51:29:20. These differences not only suggest the complementarity between multiple enrichment methods but also emphasize their collective importance in obtaining a comprehensive view of the phosphoproteome

    PNCP enrichment in endosomes and DRM fractions.

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    <p>(<b>A</b>) The most highly phosphorylated PNCPs that were present in endosome fractions from two or more cell lines, graphed as in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004130#pcbi.1004130.g001" target="_blank">Fig 1</a> except node size and color intensity represents total phosphorylation in endosome fractions. (<b>B</b>) Enrichment of proteins in endosome and DRM fractions was calculated as the ratio of amounts in endosomes or DRMs vs. the average in all other fractions and samples from that cell line, graphed as a heat map. (<b>C</b>) SFK and PAG1 phosphorylation site enrichment in LAN-6 cells (left) and SK-N-BE(2) cells (right).</p

    Collaborative groups of RTKs.

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    <p>Interaction networks show proteins that co-cluster and have Spearman correlation > 0.5 (yellow edges) or are known to interact from PPI databases (grey edges) with ALK, PDGFRA, FGFR1, and IGFR1 <b>(A)</b>; EGFR, DDR2, EPHA2, EPHB3, and PDGFRB <b>(B)</b>. Only edges linked to RTKs are shown. Proteins are grouped by the number of interactions with RTKs (<i>e</i>.<i>g</i>. the group in the center containing PAG1 and BCAR1 in <b>A</b> interacts with all four RTKs). Nodes were filtered to exclude those with lowest representation in the phosphoproteomic data. Node size and color indicates total phosphorylation as in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004130#pcbi.1004130.g002" target="_blank">Fig 2</a>. Proteins that are both known to interact and have a positive correlation have two edges (<i>e</i>.<i>g</i>. ALK edges connecting FYN, SHC1, and IRS1). PAG1, FYN, and PIK3R2, the only nodes in common to both networks, are highlighted in green.</p

    Phosphorylation site clusters displayed as a co-clustered correlation networks (CCCNs).

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    <p>RTK, SFK and PAG1 phosphorylation sites were selected from the co-cluster correlation network shown in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004130#pcbi.1004130.s012" target="_blank">S11 Fig</a>, where edges represent Spearman correlations greater than 0.5 from phosphorylation sites that co-clustered from t-SNE embeddings. Edge line thickness is proportional to correlation and the number of cases in which phosphorylation sites co-clustered from different embeddings. Two separate groups containing the most highly phosphorylated RTK and SFK sites are shown. Heat maps (right) show the primary data for phosphorylation on specific sites. (<b>A</b>) The network containing phosphorylation sites ALK 1507 (the most highly phosphorylated site on ALK), FYN 420, PDGFRA 762 and LYN 508 was extended to include other PNCP phosphorylation sites that co-clustered with positive Spearman correlation; no PAG1 phosphorylation sites co-clustered with this group. (<b>B</b>) Five highly phosphorylated PAG1 sites co-clustered with phosphorylation sites on IGF1R, DDR2, EGFR, and FGFR1. Note that the conserved peptide sequence for FYN 531; SRC 530; YES1 537 was inclusively summed to both FYN 531; YES1 537 and SRC 530 in the phosphorylation site network, but exclusively summed to FYN_i in the total protein phosphorylation networks based on the presence of other FYN phosphopeptides in the same samples. By the exclusive criteria, FYN 420; LCK 394; SRC 419; YES1 426 in (A) most likely represents FYN activation; FYN 531; YES1 537 most likely represents FYN inhibitory phosphorylation; and the inclusive summing method likely over-represents the amounts of SRC inhibitory phosphorylation in (B).</p
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