Protein precipitation of individual plasma samples

Flow-through protein fractions of depleted plasma samples were precipitated with the addition of sodium deoxycholate to a final concentration of 125 µg/ml followed by 15 min incubation at 22 °C. Trichloroacetic acid was added to a final concentration of 6 %, followed by centrifugation at 12,000×g at 4 °C for 30 min. Following centrifugation, sample supernatants containing naturally occurring peptides were collected in new tubes for separate analyses. Protein precipitates were washed with ice-cold acetone, centrifuged at 12,000×g for further 10 min and pellets resuspended in 50 µl of 6 M urea in 100 mM Tris HCl (pH 7.8). Quantitation of each sample was performed by a BCA protein assay according to the manufacturer’s instructions (Thermo Scientific, BCA UK) and 80 Âµg of protein per sample was analysed (Fig. 1). Protein precipitates and naturally occurred peptides were further processed and subjected to label-free semi-quantitative liquid chromatography tandem mass spectrometry (LC–MS/MS) and PROTOMAP analysis as described below.