Endoproteinase lys c

(N-Succinimidyloxycarbonyl)tris(2,4,6-trimethoxyphenyl)phosphonium bromide (TMPP), 4-Morpholineethanesulfonic acid monohydrate (MES), N-(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES), Trimethyl ammonium bicarbonate (TMAB), and sodium phosphate dibasic (Na₂HPO₄), sodium phosphate monobasic (NaH₂PO₄), Dimethylformamide (DMF), Cathepsin D from bovine spleen, 1,4 dithiothreitol (DTT), Iodoacetamide (IAA) and NIST-IgG1-K1 monoclonal antibody were all purchased from Sigma (St Louis, MO). Peptide standards from NIST monoclonal antibody was synthesized to 99.99% purity by Biomatik Corporation (Ontario, Canada). Human K562 predigest cell extract and sequencing grade Trypsin and endoproteinase Lys-C were purchased from Promega (Madison, WI). GLP1-Fc fusion protein was purchased from Myoderm (Norristown, PA). Optima LC–MS grade acetonitrile, water, formic acid, hydroxylamine, and Gibco PBS buffer solution were all purchased from Thermofisher Scientific (Waltham, MA).

Fifty micrograms of proteins extracted from P2′ or SV were resuspended into 200 μL of buffer containing 8 M urea, 100 mM Tris⋅HCl, pH 8 (“urea buffer”) and placed onto a Pall Nanosep Omega filter (Sigma). After shaking 1 min at 850 rpm at room temperature (Eppendorf Thermomixer), samples were centrifuged during 13 min at 6,400 × g (conditions that were optimal to remove all of the liquid from the upper chamber using a TOMY Kintaro KT-24 centrifuge). After repeating these steps two times, proteins were resuspended with 200 μL of urea buffer containing 50 mM iodoacetamide and incubated in darkness for 1 h at room temperature. The alkylation was then stopped by centrifuging as above and by resuspending protein samples with 200 μL of urea buffer containing 25 mM dithiothreitol. Unfolded proteins were subsequently washed with 20 mM ammonium bicarbonate. Proteolytic enzymes were used in a ratio of 1:50 with proteins. To generate peptides, a first digestion step (“trimming”) was performed using endoproteinase lys-C (Promega) for 6 h at 37 °C, followed by a second digestion step overnight (16 to 18 h) at 37 °C using a trypsin/lys-C combination (Promega). After centrifugation as above, digested peptides were acidified with 1% trifluoroacetic acid, concentrated, and dried using an EZ-2 Elite evaporator (SP Scientific).