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. Data was exported from PEAKS suite X plus in mzxml and pepxml formats and imported into Skyline 4.0 (Pino et al., 2017) 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 (Li et al., 2018). Briefly, steps included (1) requiring at least 2 peptides per protein and 4 transitions per precursor, (2) eliminating peptides with more than 1 missed cleavage, (3) excluding uncommon PTMs, (4) creating a training set consisting of all 12 samples, after first subjecting them to iRT calibration and mProphet peak detection using Skyline, and then further refining the target list by exclusion of all peptides with dotp values lower than 0.8, (5) enforcing peptide uniqueness by protein and eliminating iRT outliers, (6) removing peptides which were not unique to a single protein, and (7) limiting to 10 the maximum number of peptides per protein. The final filter steps utilized a sample training set, which was acquired by DIA as described in the next paragraph. 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 (Abbatiello et al., 2017).