Primary human T-cell spectral library generation workflow. Main steps used in (a) isolation of human peripheral blood mononuclear cells (PBMCs), purification of ex vivo CD3+ T-cells and in vitro activation (three ex vivo CD3+ T-cell samples and one in vitro activated CD3+ T-cell sample were obtained from PBMCs extracted from four different buffy coats). (b) Proteomic sample preparation, peptide fractionation and LC-MS/MS data acquisition. (c) Generation of individual spectral libraries for separate DDA-MS dataset. (d) Generation of a combined assay library compatible for multiple SWATH-MS data analysis platforms and quality control of assay libraries.