Sample preparation for proteomic analysis
To remove high-abundance proteins, 30 µL of plasma samples were diluted 1:4 with multiple affinity removal system (MARS) buffer A (Agilent Technologies, Santa Clara, CA, USA) and filtered with 0.22 μm Spin-X filters (Corning Costar, NY, USA). Individual plasma samples were depleted of six high-abundance human plasma proteins [albumin, Immunoglobulin (Ig) G, IgA, transferrin, haptoglobin, and antitrypsin) using a MARS column (Hu-6HC, 4.6 × 100 mm, Agilent Technologies, Santa Clara, CA, USA) on an Agilent 1260 HPLC system. Depleted plasma samples were concentrated by centrifugal filtration using a 3 kDa Amicon filter (Millipore, Burlington, MA, USA). Protein concentration was measured using the Bicinchoninic acid (BCA) assay.For protein digestion, 100 µg of each sample was precipitated by adding a 5-fold volume of ice-cold acetone prior to digestion. The dried samples were reconstituted in 50 µL of SDT buffer (2% sodium dodecyl sulphate, 0.1 M dithiothreitol in 0.1 M Tris HCl pH 8.0). The denatured proteins were heated at 95 °C, and subsequently digested using a filter-aided sample preparation (FASP) method, as previously described [