Samples were analyzed by both liquid chromatography – selected reaction monitoring (LC-SRM) and liquid chromatography – data dependent acquisition (DDA) tandem mass spectrometry (LC-MS/MS) as previously described46 (link),48 (link). Global analysis was performed on an Orbitrap – Velos coupled with an Eksigent 2D nano-LC, while targeted (LC-SRM) analysis was performed on a Qtrap 5500 coupled with a Dionex Ultimate 3000 UHPLC utilizing optimized conditions described previously. LC-SRM data was directly loaded into Skyline and transition quality, peak shape, and peak boundaries were manually validated. Resulting integrated peak areas were directly exported and protein quantity (pmol/g) was calculated against the known spike of stable isotope labeled peptides. LC-MS/MS data was queried against the SwissProt human database using Mascot (v2.3.1) and directly loaded into Scaffold™ (Proteome Software). Peptide Spectral Matches (PSMs) were directly exported with a 99% confidence in protein identifications and at least 2 unique peptides per protein, resulting in a false discovery rate of 0.54%. Statistical analysis for proteomics data, including principal component analysis, was performed using the MetaboAnalyst (v3.0) software suite49 (link).
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