This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. In data-independent acquisition (DIA), MS/MS spectra are acquired continuously at predetermined m/z windows independent of peptide precursor detection. Ideally, DIA would cover the entire mass range with small precursor m/z isolation windows and a total cycle time of <5 sec to ensure adequate chromatographic sampling. Unfortunately, current ion trap mass spectrometers are limited to 30-50 MS/MS scans in a 5-second cycle. Thus, we need to perform many runs to cover the mass range, increase isolation window size, or cover an informative subset of the m/z range. We developed methods to deconvolute DIA fragmentation spectra derived from multiple peptides into their individual components. We empirically optimized fragmentation window selection in Saccharomyces cerevisiae and Caenorhabditis elegans maximizing protein identifications in a run.