Proteome purify 12 human serum protein immunodepletion resin

High abundant plasma proteins were depleted using the Proteome Purify 12 Human Serum Protein Immunodepletion Resin (R&D Systems, Minneapolis, MN, USA). Briefly, 10 μL of plasma was mixed with 1 mL of immunodepletion resin for 60 min. The mixture was transferred to Spin-X filter units and centrifuged. The protein concentration of the resulting eluate was determined using the Pierce™ 660 nm protein assay (Thermo Fisher Scientific). Protein digestion was performed using the FASP protocol, as described above. A total of 10 μg of protein was digested at a 1:25 enzyme-to-protein ratio. The tryptic digest was acidified at a 1:10 ratio using 2% TFA, 20% ACN.

Each of the six sample pools were immunodepleted using Proteome Purify 12 Human Serum Protein Immunodepletion Resin (R&D Systems, Minneapolis, MN, USA; #IDR012). Briefly, 45 µL of serum pool was incubated with 4.5 mL of immunodepletion resin for 45 min on a rotator at 4 °C in polypropylene columns (Thermo Scientific Pierce, Waltham, MA, USA; #PI29924). Following incubation, the depleted serum was collected and concentrated to 500 µL using 5 kDa molecular weight cut-off Vivaspin columns (Sartorius, Tagelswangen, Switzerland; #VS04T11). Samples were vacuum dried and resuspended in a buffer consisting of 100 mM triethylammonium bicarbonate (TEAB) and 0.1% SDS, and the protein content was determined using the Coomassie Plus Assay Kit (Thermo Scientific Pierce, Waltham, MA, USA; #90064). A quantity of 100 µg of protein from each pool at 1 µg/µL was taken for tryptic digestion followed by TMT labelling.