Proteominer protein enrichment large capacity kit

The ProteoMiner Protein Enrichment Large Capacity Kit (Bio-Rad Laboratories, Hercules, CA, USA) was used for the enrichment of low-abundance proteins, following the manufacturer’s instructions. Briefly, serum was added to spin columns containing the beads and incubated at room temperature for 2 h with constant end-to-end rotation. After washing the columns, the proteins bound to the beads were eluted with the appropriate buffer, divided into aliquots, and stored at −80 °C until use. The protein concentration of each sample was assessed using an assay based on the Bradford method. dl-Dithiothreitol (Sigma-Aldrich) was added (final concentration 10 mM) and the mixture was incubated at 37 °C for 1 h. Iodoacetamide (Sigma-Aldrich) was added (final concentration 10 mM) and the mixture was incubated 45 min in the dark, followed by centrifugation (14000g, 15 min). The sample was diluted 4 times by adding 25 mM ammonium bicarbonate buffer. Trypsin (trypsin protein = 1:50) was then added and incubated at 37 ℃ overnight. The tryptic peptides were then acidified with formic acid (FA) to achieve pH < 3 before desalted using Ziptip C18 (Millipore Corp., Billerica, MA, USA). The desalted peptides were then dried under vacuum.

To reduce the concentration of high-abundance proteins and increase the concentration of low- and medium-abundance ones, serum samples were processed by the ProteoMiner Protein Enrichment Large-Capacity Kit (Bio-Rad, Hercules, CA, USA) in accordance with the manufacturer’s instruction. Depleted samples were then precipitated with 4 volumes of ice cold acetone at −20 °C for 2 h and centrifuged for 60 min at 20,000× g at 4 °C. Protein pellets obtained were dissolved in a lysis buffer containing 7 M urea, 2 M thiourea, 4% w/v 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS), 1% w/v dithiothreitol (DTT), 0.2% w/v 3–10 carrier ampholytes, and 2 mM tributylphosphine. The protein concentration was determined using a modified Bradford assay (Bio-Rad) according to the manufacturer’s instructions.