Identification of Substrates of MBL Associated Serine Protease-1 (MASP-1) from Human Plasma using N-terminomics Strategy
Human blood was drawn from healthy adult volunteers after taking their informed consent. The study was approved by the institutional ethics committee, Ref. No. All India Institute of Medical sciences (AIIMS) /Patna/Institutional Ethics Committee/2019/413. All the experimental work was done in accordance with the relevant guidelines and regulations. Acid-citrate-dextrose (ACD) was used as an anticoagulant (6 vol. of blood to 1 vol. of ACD). Platelet-rich plasma was obtained by centrifuging the ACD blood at 200 g for 15 min. This was followed by plasma depletion of albumin and Immunoglobulin G (IgG) using antibody-based Abundant Protein Depletion Spin Columns (Pierce, Thermo Fisher, MA, USA). The antibodies were specific to the Fc domain of immunoglobulins.
Steps to reproduce
A transamination reaction was performed as described previously (Bhagwat et al., 2020; Sonomura et al., 2009). Buffer exchange was performed with 50 mM Ammonium bi Carbonate (ABC) in 2 M Urea , in 100 mM phosphate buffer pH 6.0. Disulfide bonds were reduced using 5 mM Dithiothreitol and the sample was incubated at 56ºC for 45 min. The reaction mixture was subjected to carboxymethylation using Iodoacetamide (IAA) to a final concentration of 14 mM. The protein mixture was then incubated at 25ºC for 30 min in dark. The unreacted IAA was quenched using 0.5 M DTT and incubation in the dark for 15 min. Buffer exchange was repeated with 50 mM ABC without urea. The protein sample was digested by rMASP-1 (1:40) and the mixture was incubated at 25ºC for 16 hrs. Guanidination was performed by increasing the pH of the reaction to 10.3. Methylisourea 0.5 M was then added to the reaction mixture and incubated at 4ºC for 20 hr. The reaction was stopped by adding 0.1% Trifluoroacetic acid. The pH of the reaction mixture was adjusted to pH 8.0. Biotin 5 mM added to the reaction mixture and the protein sample was incubated overnight at 4ºC. Streptavidin agarose column was initially washed with 100mM phosphate buffer in 0.15M NaCl, pH 7.2. The biotinylated peptides were eluted using 50mM glycine HCl, pH 2.7. The pH of the eluted peptide mixture was increased to 8.0 and digested with Trypsin at 37ºC overnight. The peptide mixture was lyophilized and reconstituted in 10% formic acid for mass spectrometry analysis. The samples were run on an Orbitrap Fusion Tribrid Mass Spectrometer. The peptide mixture was separated using a C18 analytical column (500 mm × 0.75 mm i.d., 2.0 μm particle size) using an isocratic gradient of buffer A, 95% acetonitrile 10% buffer A, and 0.1% formic acid, over a period of 60 min at a flow rate of 300 µl/min. MS-MS data were acquired using the Data Dependent Acquisition (DDA) protocol (50-2500 m/z) in positive ion mode. The human database was obtained from Uniprot having revised Swiss-Prot entries (release – 2017, 20,341 entries). The parameters for precursor and fragment ions were set as maximal mass deviation as 7 and 20 ppm, respectively. Oxidation (Met), N-acetylation and guanidination were chosen as the variable modifications. Carbamidomethylation (Cys) was set as fixed modification. The digestion mode was selected to identify peptides with a minimum length of 7 amino acids. The peptide mass tolerance was set at ±20 ppm, fragment mass tolerance at 0.1 Da, and trypsin digestion with missed cleavages up to 2 were allowed. All data were reported based on 99% confidence for protein and peptides identification as determined by false discovery rate (FDR) ≤ 1%. The spectral data were analyzed using Proteomic Discoverer software.