Raw spectral library and DIA assay library construction
An O. mossambicus gill spectral library was generated using the peptide-to-spectrum matches and protein annotations from DDA data as previously described [30] to create a non-redundant raw library of MS2 spectra. The initial target list of proteins was filtered using multiple QC criteria to reduce the number of transitions, precursors, peptides, and proteins to a unique set which provided the highest diagnostic value for quantitation. Transitions for the initial target list were chosen automatically from library spectra using Skyline transition settings based on the following criteria: ion 3 to last ion -1; fragment ion charge 1; precursor charge range 1 – 5; MSMS mass accuracy threshold within 20 ppm of the expected mass. This initial target list was filtered in seven sequential steps using Skyline following a previously published method [31]. The complete assay library including all relevant metadata and corresponding data for the sample training set is available at Panorama Public (https://panoramaweb.org/lr03.url). This assay represents a tier two assay [32].