Trypsin porcine pancreas

The chemicals and reagents used in this work are the following: water (LC-MS grade-LiChrosolv^®) (Merck KGaA, Darmstadt, Germany), acetonitrile (LC-MS grade-LiChrosolv^®) (Merck KGaA, Darmstadt, Germany), 2-propanol (LC-MS grade-LiChrosolv^®) (Merck KGaA, Darmstadt, Germany), ammonium bicarbonate (NH₄HCO₃) (Sigma-Aldrich, Buchs, Switzerland), RapiGest^TM SF surfactant (Waters, Milford (MA), USA), DL-dithiothreitol (DTT) (Sigma-Aldrich, Buchs, Switzerland), iodoacetamide (IAA) (Sigma-Aldrich, Buchs, Switzerland), trypsin (porcine pancreas) (Sigma-Aldrich, Buchs, Switzerland), trifluoroacetic acid (TFA) (Honeywell SC, Seeelze, Germany), formic acid (FA) LiChropur^® (Merck KGaA, Darmstadt, Germany), Pierce^TM HeLa protein digest standard (Thermo Scientific^TM, Waltham, MA, USA) and MMI-L low concentration tuning mix (Agilent Technologies, Santa Clara, CA, USA).

Proteases used were Chymase (Sigma C8118), Cathepsin D (Sigma SRP6415), Cathepsin G (RP-77525), Cathepsin E (Biovision 7842), Pepsin (Roche 10108057001), Trypsin porcine pancreas (Sigma T0303-1G), Lysozyme from chicken egg white (Sigma L6876), Napsin A (RND 8489-NA). Digestion experiments were carried out with purified human hemoglobin (Sigma H7379) and recombinant or purified proteases. 100 μg hemoglobin (ca. 1.56 nmol) were digested with Chymase (in Tris-HCl 0.05 M, pH 8.0/0.26 M NaCl), Cathepsin D, G and E (in 0.2 M citrate buffer, pH 5.0), Pepsin (in 20 mM sodium acetate buffer, pH 3.5), Trypsin (in 0.1 M Tris–HCl, with 10 mM CaCl2, pH 8), Lysozyme (in 10 mM Tris-HCl, pH 8) or Napsin A (in 0.2 M NaCl, 0.1 M sodium acetate, pH 3.6). All proteases were used at a 1:100 molar ratio (15 pmol) and reactions were incubated at 37°C for 2 h.

Trypsin (porcine pancreas, Sigma-Aldrich) activity was measured at 25°C in a reaction mixture that contained from 0 to 81.6 mM leupeptin or pseudospumigin A and the hydrolysis was followed at every 2 min for 1 h, as described earlier (Liu et al., 2015 (link)).