2 4 morpholino ethanesulfonic acid mes

N-Fluorenylmethyloxycarbonyl (Fmoc) protected amino acids, amino acid attached 2-chlorotrityl resins, 1-[bis(dimethylamino) methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluoroph osphate (HATU), and 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) were purchased from Chem-Impex International, Inc. (Wood Dale, IL) and AAPPTEC LLC (Louisville, KY). Potassium hexacyanoferrate (II) trihydrate, potassium nitrate, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), N-hydroxysulfosuccinimide (Sulfo-NHS), 6-mercapto-1-hexanol, and 6-mercapto-1-hexanoic acid were obtained from Sigma-Aldrich (St. Louis, MO). Dithiothreitol (DTT), sodium phosphate dibasic heptahydrate (Na₂HPO₄•7H₂O), sodium phosphate monobasic (NaH₂PO₄) and 2-(4-morpholino)ethanesulfonic acid (MES) were purchased from Fisher Scientific (Hampton, NH). Purified recombinant human cathepsin B (~60% 37 kDa inactive form and ~40% 25 kDa active form) was acquired from R&D Systems Inc. (Minneapolis, MN). Cathepsin B solutions were activated by incubation in 25 mM MES buffer (pH = 5.0) containing 5 mM DTT for 15 min before proteolysis experiments.

Chloroform and ethanol (HPLC grade) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Methanol was LC-MS grade from Fisher Scientific (Pittsburgh, PA, USA). 2-(4-Morpholino)ethane sulfonic acid (MES) was BioReagent grade also from Fisher Scientific (Pittsburgh PA, USA). Water was purified by a milliQ system (EMD Millipore, Billerica, MA, USA). 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) was purchased from Thermo Scientific/Pierce Biotechnology (Rockford, IL, USA). Phospholipase C from B. cerus, ammonium acetate, and 2,5-dihydroxybenzoic acid (DHB) were also purchased from Sigma-Aldrich (St. Louis, MO, USA).