Coomassie plus assay kit

The Per a 5 gene was subcloned into pFastBac1 vector (Novagen, Madison, WI, USA) using EcoR I and Sal I sites and the resulted construct was transformed into E. coli strain DH10Bac to generate recombinant bacmid. The positive colonies were selected and followed by PCR identification. The recombinant bacmid was transfected into Sf-9 cells by using Cellfectin (Invitrogen Corporation, Carlsbad, USA) and incubated in SF-900II liquid medium (Invitrogen Corporation, Carlsbad, USA) for 5 days at 27°C until the cells got swollen. The supernatant was collected as P1 viral stock. P2 viruses were amplified for later infection. A total of 500 mL of Sf-9 cells were infected by P2 viruses and harvested at 72 h. The cells were lysed against 50 mM Tris-HCl with 300 mM NaCl and 5% glycerol. The supernatant was loaded on Ni-NTA column (Genscript, Nanjing, China), washed with running buffer containing 50 mM Tris-HCl, 300 mM NaCl, and 5% glycerol (pH 8.0) and eluted with elution buffer containing 50 mM Tris-HCl, 300 mM NaCl, 250 mM imidazole, and 5% glycerol (pH 8.0). The eluted fractions were obtained and identified as Per a 5 (iPer a 5). The purified iPer a 5 was dialyzed in carbonate-bicarbonate buffer (0.05 M, pH 9.6) for further investigation. The concentration of iPer a 5 was determined by using a Coomassie Plus assay kit with BSA as standard (Thermo Scientific Pierce, Rockford, IL, USA).

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.

For ferritin and protein determination, the cells were thawed and then centrifuged at 13,000× g for 15 min at 4 °C to discard the cellular debris from the proteins contained in the supernatant. The ferritin content was determined by the Human Ferritin ELISA Kit (Thermo Scientific) and was measured using a microplate reader (infinite 200 pro Tecan i-control, Switzerland) at 450 nm with a reference wavelength set at 550 nm according to the kit instructions. The Coomassie Plus Assay Kit (Thermo Scientific) was used to determine the total protein content at 595 nm by the microplate reader. The determination of the latter was necessary to normalize the ferritin concentration.