Filtering of the raw spectral library and final composition the DIA assay library
We generated a raw MS2 spectral library from DDA data of all twelve samples, which contained 7004 proteins, 82605 peptides, 90350 precursors (different charge states of peptides), and 462868 transitions (fragment ions) (Figure 1). 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; 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, Levitan, Gomez-Jimenez & Kültz, 2018). Briefly, steps included (1) requiring a minimum number of 2 peptides per protein and 4 transitions per precursor, (2) elimination of peptides with more than 1 missed cleavage, (3) exclusion of PTMs other than Met/ Pro oxidation, Cys carbamido-methylation, and protein N-acetylation, (4) creation of a training set consisting of all 12 samples, which were first subjected to iRT calibration and mProphet peak detection using Skyline, and then further refined by excluding all peptides with dotp values less than 0.8 in all samples (5) enforcing peptide uniqueness by protein and eliminating iRT outliers (6) removing all repeated peptides to represent each protein by unique peptides, and (7) limiting the maximum number of peptides per protein to 10. Applying this stringent set of QC filter steps resulted in a final DIA assay library target list containing 52,361 transitions, 9226 precursors, 9226 peptides, and 2120 proteins (Figure 1). The five most common transitions (fragment ions) included in this kidney DIA assay library are y4 to y8 ions, followed by b4, b5, y3, and y9 ions (Figure 1e). Despite requiring each protein to be represented by at least two peptides in the raw spectral library, one-third (711) of the DIA assay library proteins are only represented by a single peptide (although still by multiple transitions) (Figure 1f). The reason is that at least one peptide was excluded by DIA assay filter steps outlined above because it did not meet one of the QC criteria.