Optimising sample preparation of the plasma peptidome

We further expanded on previous optimizations to isolate the plasma peptidome and compared various methods to maximize identifications with speed and ease. Previous studies have reported loss of polypeptides binding to high abundant proteins during depletion strategies. We hypothesized that rapid chaotropic denaturation of plasma with urea under reducing conditions would liberate non-covalently bound peptides to improve recovery during protein depletion. We also compared depletion strategies to isolate the peptidome including protein precipitation and removal with either TCA, acetone or acetonitrile (AcN). Following centrifugation of precipitated proteins, the supernatant containing peptides was collected. For acetone and AcN precipitations, an additional vacuum centrifugation step was required followed by resuspension of peptides in aqueous buffer. Our comparison of peptidome isolation also included removal of proteins with size-exclusion 10 kDa MWCO filters. All peptide isolations were acidified to 0.1% TFA, adjusted to 5% acetonitrile and desalted with HLB-SPE. Peptides were analysed by single-shot nanoUHPLC-MS/MS employing both HCD and EThcD and quantified by LFQ