Experimental workflow for proteomics experiments. (A) Pure platelet preparations were obtained from whole blood by using a multistep approach (see “Materials and methods”), and lysates were subjected to affinity capture of PtdIns(3,4,5)P₃-binding proteins by using PtdIns(3,4,5)P₃-coupled beads. Eluate sample complexity was reduced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis separation of proteins, followed by trypsin digest, nano-HPLC, and MS analysis. Data were subject to stringent filtering and analysis using a range of bioinformatics tools. (B) Human platelet lysates were incubated with control (Ctl) or PtdIns(3,4,5)P₃ [PIP₃, or after preincubation with competing free PtdIns(3,4,5)P₃ (PIP₃+)]-coupled beads for 90 minutes at 4°C before washing, elution, and analysis by SYPRO Ruby gel staining. (C) Experiments conducted as described in panel B were subjected to liquid chromatography–tandem mass spectrometry (LC-MS/MS) analysis. Histograms demonstrate validation of the proteomics approach by quantitative analysis of known PtdIns(3,4,5)P₃-binding proteins with the T3PQ method across the independent donors. Bars represent the mean of 3 independent donors + standard error of the mean. ACD, acid citrate dextrose; Ctl, control; HT, HEPES-Tyrode’s buffer; PRP, platelet-rich plasma.