Figure 3. DIA analysis of HLA DR and DQ peptidomes of Maver-1 and DOHH2 cells.

(A) Precursor mass distribution from a triplicate DDA analysis of the Maver-1 HLA DR peptidome. The m/z distributions of all peptides with charge state 2 (z=2, solid black), 3 (z=3, solid grey), 4 (z=4, dotted grey) and 5 (z=5, dotted black) were plotted. (B) Library generation and DIA analysis of Maver-1 and DOHH2 cells. Spectral libraries were created using SpectraST based on SEQUEST results filtered to 1% FDR with Percolator from triplicate DR and DQ DDA analyses of Maver-1 and DOHH2 cells. DR and DQ peptidomes from Maver-1 and DOHH2 cells were acquired in DIA mode in triplicates and analysed using Skyline. The graph displays the number of unique entries in the spectral libraries (light grey) and the number of peptides identified in a triplicate DIA analysis with an mProphet q-value below 0.01 (dark grey). (C) Reproducibility of Maver-1 and DOHH2 DIA peptidome analysis. Triplicate DIA DR and DQ peptidome analyses of Maver-1 and DOHH2 cells were analysed using Skyline and filtered to 1% FDR with mProphet. Euler-Venn diagrams display the overlap of peptide identifications between replicates. (D) Comparison of sensitivity between DDA and DIA with varying input cell numbers. DR peptidomes were isolated from Maver-1 lysate corresponding to 1, 5, 10, 25 and 100 million Maver-1 cells. Samples were acquired in either DDA or DIA mode in triplicates. DDA data was analyzed with SEQUEST and results filtered for 1% FDR with Percolator. DIA data was analyzed with Skyline using the Maver-1 DR library described in (B) and the FDR was estimated with mProphet and set to 1% at peptide precursor level. The graph shows the means and corresponding standard deviations of unique peptides identified in DDA mode (dark grey) and DIA mode (light grey) for the different input amounts.