Endoproteinase lys c

Cell conditioned media were thawed on ice and were concentrated down to ∼100 μl using Amicon Ultra-15 3k molecular weight cut off (MWCO) centrifugal filter units (Millipore Sigma). Buffer exchange into PreOmics iST lysis buffer was performed, followed by incubation at 95 °C for 10 min for reduction and alkylation of proteins. Protein amounts were normalized using BCA assay (Thermo Fisher Scientific), and equal amounts from each condition were subjected to enzymatic cleavage for 3 h by adding equal amounts of endoproteinase Lys-C and trypsin (ThermoFisher Scientific, # A40009) in a 1:50 (wt/wt) enzyme:protein ratio. De-salting and purification were performed according to the PreOmics iST protocol on a styrene divinylbenzene reversed-phase sulfonate sorbent. Purified peptides were vacuum-centrifuged to dryness and reconstituted in double-distilled water with 2 vol% ACN and 0.1 vol% FA for single-run LC-MS analysis.

Purified SARS-CoV-2 spike antigen (S1-RBD) [His-Tag (HEK293)] was purchased from the Native Antigen Company [REC31882-500].
Tris-HCl was from Invitrogen. Dithiothreitol (DTT), iodoacetamide (IAM) and urea were from Sigma-Aldrich, digestion enzymes endoproteinase Lys-C and chymotrypsin from Thermo Scientific and MS grade formic acid and acetonitrile from Biosolve.

Microsomal proteins were predigested for 3 hours with endoproteinase Lys-C (0.5 μg/μl; Thermo Scientific) at 37 °C in 6 m urea 2 m thiourea, pH 8 (Tris-HCl). After 4-fold dilution with 10 mm Tris-HCl (pH 8), samples were digested with 4 μl Sequencing Grade Modified trypsin (0.5 μg/μl; GE Life Sciences) overnight at 37 °C. The reaction was stopped by addition of trifluoroacetic acid (TFA) to reach pH ≤ 3 before further processing.