2-fold improved coverage of HLA immunopeptidomes with up to 15,000 distinct HLA-I and HLA-II peptides from 4e7 cells. Our optimized single-shot MS acquisition method on the SCP maintains high coverage, eliminates the need for biochemical fractionation and reduces input requirements to 1e7 cells for > 800 distinct HLA-I peptides. Moreover, we find that the binding motifs of specific HLA alleles can influence peptide fragmentation patterns, and that optimized collision energies can result in complementary b/y ion sequence coverage. Our optimized single-shot SCP acquisition methods enable sensitive, high throughput and reproducible immunopeptidome profiling and the detection of peptides from non-canonical open reading frames and clinically relevant cancer-testis antigens from less than 4e7 cells or 15 mg wet weight tissue. ]]>