Identification of protein phosphorylation events initiating GPVI platelet activation. (A) Workflow summary for multiplexed, quantitative phosphoproteomics analysis of resting vs CRP-XL-activated platelets. Blood was drawn from n = 5 healthy human donors to prepare washed platelets (>99.9% CD41⁺ by flow cytometry). Platelets were incubated with apyrase, indomethacin, and Integrilin to inhibit feedback processes that potentiate platelet activation downstream of GPVI (condition 1). Platelet samples were each equally divided into control vs stimulated (+10 μg/mL CRP-XL) samples for 5 minutes, as a range of GPVI-mediated intracellular signaling events associated with platelet function are known to be detectable at this time point under these conditions.^82-84  After 5 minutes, platelet samples were separately lysed, digested, and alkylated before phosphopeptide enrichment on anti-pTyr resin and TiO₂ beads and addition of 10-plex tandem mass tag (TMT) labels. Ten pTyr- and 10 TiO₂-enriched samples were each separately combined for 2 10-plex analyses of phosphopeptides on an Orbitrap Fusion MS. (B) Example MS2 spectra of tryptic peptide and ion fragmentation identifying phosphorylation of PLCγ2 Y₇₅₉ and MS3 quantitation of phosphopeptide reporter ion intensities from (n = 5) control (gray) vs +CRP-XL (black) platelet samples. (C) Principal component analyses of all identified phosphopeptides from control (orange) and +CRP-XL (blue) samples following pTyr (top) and TiO₂ enrichment (bottom) and multiplex MS analysis. (D) Scatter plots of measured mean phosphopeptide reporter ion intensities from control vs +CRP-XL samples following pTyr (left) and TiO₂ enrichment (right). Significant changes colored by Benjamini–Hochberg false discovery rate (FDR), as indicated. (E) Volcano plots of phosphopeptide reporter ion intensity ratios (−log₂ +CRP-XL/control) vs P values. Phosphopeptides with highly significant differential intensities (FDR < 0.01) shaded by FDR, as indicated. A total of 204 (120 increasing, 84 decreasing) and 979 (619 increasing, 360 decreasing) significantly differential phosphopeptides were identified in control vs +CRP-XL samples following pTyr and TiO₂ enrichment, respectively. (F) Reporter ion intensity measurements of representative phosphopeptides corresponding to specific protein phosphorylation sites for n = 5 control (yellow) and +CRP-XL (green) samples. (G) Lysates of control and +CRP-XL platelet samples above were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and examined by western blot with phosphorylation site-specific antisera (phos.) against PLCγ2 Y₇₅₉, DAPP1 Y₁₃₉, GSK3α S₂₁, Hsp27 S₈₂, VASP S₂₃₉, and FLNA S₂₁₅₂. Blot pixel intensity measurements (a.u.) are shown for n = 3 samples. Western blot for α-tubulin serves as a control for total protein levels. Significance (*P < .1, **P < .05, ***P < .01) determined by paired Student t test. Note: Interactive and searchable versions of the phosphoproteomics data and graphs above are available at: https://kcvi.shinyapps.io/STARTapp_359-pTyr/ and https://kcvi.shinyapps.io/STARTapp_359-TiO2/.