Pierce top 12 abundant protein depletion spin columns kit

Firstly, the cellular debris of plasma sample was removed by centrifugation at 12,000 g at 4°C for 10 min. Then, the supernatant was transferred to a new centrifuge tube. The top 12 high abundance proteins were removed by Pierce™ Top 12 Abundant Protein Depletion Spin Columns Kit (Thermo Fisher). Finally, the protein concentration was determined with BCA kit according to the manufacturer’s instructions.

The cellular debris was removed from serum samples by centrifugation at 12,000g, 4°C for 10 min. The supernatant was collected and treated using the Pierce™ Top 12 Abundant Protein Depletion Spin Columns Kit (Thermo Fisher Scientific, IL, USA) to remove the 12 high-abundance proteins. Protein quantification was performed using a BCA kit (Beyotime Institute of Biotechnology, Hangzhou, China) according to the manufacturer’s instructions. Before digestion, the protein solution was reduced with dithiothreitol (5 mM, at 56°C for 30 min) and alkylated with iodoacetamide (11 mM, at room temperature for 15 min, in darkness). Then, the solutions were diluted with tetraethyl ammonium bromide (TEAB; 100 mM) to urea concentration less than 2 M. Finally, proteins were digested using two-step digestion with trypsin (Promega, Madison, WI, USA), 1:50 trypsin-to-protein mass ratio overnight and 1:100 trypsin-to-protein mass ratio for 4 h. For TMT labeling, digested peptides were desalted using a Strata X C18 SPE column (Phenomenex, Torrance, CA, USA), vacuum-dried, dissolved in TEAB, and then prepared using labeling reagents for 2 h at room temperature according to instructions provided by a TMT kit (Thermo Fisher Scientific). Samples were dried by vacuum centrifugation.

Relative proteins were quantified using Tandem Mass Tag mass spectrometry by PTM BIOLABS company. Firstly, the top 12 high abundance proteins were removed from serum samples by Pierce Top 12 Abundant Protein Depletion Spin Columns Kit (Thermo Fisher Scientific, Waltham, MA, USA). Trypsin was then used for the protein digestion. After trypsin digestion, peptide was desalted by Strata X C18 SPE column (Phenomenex, CA, USA) and labelled according to the manufacturer’s protocol for TMT kit/iTRAQ kit (Thermo Fisher Scientific, Waltham, MA, USA). Then, the tryptic peptides were fractionated into fractions by high pH reverse-phase HPLC using Thermo Betasil C18 column. The peptides were subjected to NSI source followed by tandem mass spectrometry (MS/MS) in Q ExactiveTM Plus (Thermo Fisher Scientific, Waltham, MA, USA) coupled online to the UPLC. Finally, the resulting MS/MS data were processed using Maxquant search engine (v.1.5.2.8). Tandem mass spectra were searched against the Uniprot database concatenated with reverse decoy database.