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].