The Use of Variable Q1 Isolation Windows Improves Selectivity in LC–SWATH–MS Acquisition

As tryptic peptides and metabolites are not equally distributed along the mass range, the probability of cross fragment ion interference is higher in certain windows when fixed Q1 SWATH windows are applied. We evaluated the benefits of utilizing variable Q1 SWATH windows with regards to selectivity improvement. Variable windows based on equalizing the distribution of either the precursor ion population (PIP) or the total ion current (TIC) within each window were generated by an in-house software, swathTUNER. These two variable Q1 SWATH window strategies outperformed, with respect to quantification and identification, the basic approach using a fixed window width (FIX) for proteomic profiling of human monocyte-derived dendritic cells (MDDCs). Thus, 13.8 and 8.4% additional peptide precursors, which resulted in 13.1 and 10.0% more proteins, were confidently identified by SWATH using the strategy PIP and TIC, respectively, in the MDDC proteomic sample. On the basis of the spectral library purity score, some improvement warranted by variable Q1 windows was also observed, albeit to a lesser extent, in the metabolomic profiling of human urine. We show that the novel concept of "scheduled SWATH" proposed here, which incorporates (i) variable isolation windows and (ii) precursor retention time segmentation further improves both peptide and metabolite identifications.