Overview of the analytical strategy. (A) Platelets were isolated from fresh blood donations and lysed, and proteins were carbamidomethylated and digested using trypsin. (B) Quantitative analysis was conducted in a 2-pronged way. (1) For absolute quantification of protein copy numbers, equal amounts of digest from the 4 donors were combined and peptides separated by in-solution isoelectric focusing. Obtained fractions were analyzed by LC-MS/MS on an LTQ Obritrap Velos and copy numbers were calculated based on the NSAF method. (2) For quantifying the biologic variance of the human platelet proteome, 100 μg of each sample were labeled with iTRAQ 114, 115, 116, and 117, respectively. Samples were multiplexed and fractionated using different techiques, namely SCX, IEF, HILIC, and COFRADIC. Obtained fractions were analyzed by LC-MS on Orbitrap XL and Qstar Elite mass spectrometers. (C) To increase the coverage of the human platelet proteome, a 2-step TiO₂ enrichment, first with low and then with high specificity for phosphopeptides was conducted and samples analyzed by LC-MS/MS on a q-Exactive mass spectrometer.