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  • For each of the rHSA samples approximately g

    2018-11-07

    For each of the rHSA samples, approximately 10µg were diluted in 50mM ammonium bicarbonate to a total volume of 200µl. To each solution 10µl of 250mM dithiothreitol (DTT) in 50mM NH4HCO3 were added followed by incubation for 1h at 60°C. Next, 20µl of 250mM iodoacetamide in 50mM NH4HCO3 were added and samples were incubated at room temperature in the dark for 30min. The alkylation reactions were quenched by the addition of an additional 20µl of 250mM DTT and the samples were split into 2×125µl aliquots and transferred onto 10kDa MWCO spin filters. Samples were centrifuged for 20min at 14,000×g then washed with 2×200µl of 50mM NH4HCO3 in the same manner. Trypsin and chymotrypsin solutions were prepared by adding 1000µl of 50mM NH4HCO3 to 20µg and 25µg lyophilized protein, respectively. One 125µl aliquot from each sample was digested with trypsin and the other with chymotrypsin. Digestion was carried out by spinning 100µl of trypsin kinase inhibitors solution through the filters at 10,000×g over 20min, then spinning 100µl of 50mM NH4HCO3 through the filter under the same conditions. The flow through from the digestion steps was collected in clean tubes and evaporated to dryness in a vacuum centrifuge then re-suspended in 40µl of injection solvent (3% acetonitrile, 0.2% formic kinase inhibitors and 0.05% TFA in water) prior to LC–MS/MS analysis.
    LC–MS analysis—Sample analysis For each sample, triplicate 2µl aliquots were analyzed by loading onto a Waters Symmetry C18 trap column (180µm×20mm with 5µm beads) and desalting with 0.1% formic acid in water (solvent A) for 3min at 5.0µl/min before separating on a Waters nanoAcquity UPLC BEH130 C18 reverse phase analytical column (100µm×100mm with 1.7µm beads). Chromatographic separation was achieved at a flow rate of 0.500µl/min over 70min in six linear steps as follows (solvent B was 0.1% formic acid in acetonitrile): Initial—3% B, 2min—10% B, 40min—30% B, 50min—95% B, 55min—95% B, 56min—3% B, final—3% B. The eluting peptides were analyzed by MS and MS/MS using a Waters Synapt HDMS system operating in data directed acquisition (DDA) mode. MS survey scans were 1s in duration and MS/MS data were collected on the four most abundant peaks until either the total ion count exceeded 3000 or until 3s elapsed. Within each analysis, redundant analyses were limited by excluding selected peaks ±1.15 mass-to-charge (m/z) for 60s. Between triplicate analyses, previously selected peaks were prevented from being reanalyzed by using m/z (±1.15) and retention time (±60s) as exclusion criteria. Peaks from singly-charged peptides were also excluded from selection for MS/MS analysis. The instrument was calibrated prior to sample analysis using the fragmentation products of [Glu1]-Fibrinopeptide B. Calibration accuracy was maintained throughout the analyses using a nano-lock spray of 100fmol/µl [Glu1]-Fibrinopeptide B, which was sampled for 1s once every 30s. The lock mass correction was applied to the data during processing.
    LC–MS analysis—Qualitative data processing Data were qualitatively analyzed using the Mascot software package, available from Matrix Science Ltd. (Boston, MA, U.S.A.). The raw data were processed using Mascot Distiller (version 2.4.2) to create Mascot Generic Files (MGFs) and database searches were performed using Mascot (version 2.4), against the human protein entries in the 2013_01 UniProtKB/Swiss-Prot database. MGF files from the triplicate analyses of both the trypsin and chymotrypsin digests were combined and submitted as a single search for each sample. Peptide and MS/MS mass tolerances were 100ppm and 0.1Da, respectively, and semi-tryptic and semi-chymotryptic peptides, from 2+ to 5+ charge state and having up to three missed cleavages, were considered. Carbamidomethylation of cysteine was specified as a fixed modification and oxidation of methionine and hexose addition on lysine and arginine were considered as variable modifications. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD001248 and DOI 10.6019/PXD001248.