Human NE, PR3, and CG were purchased from Elastin Products Company. Reactions were performed in 50 ml volumes containing 15 mg His 6 -proHNP1 and 2 mg of protease. Buffer for NE and PR3 reactions was 50 mM Tris-HCl pH 7.5, 150 mM NaCl and CG reactions were performed in 50 mM Tris-HCl pH 8.3, 150 mM NaCl [32] . Incubation was performed at 37uC for 18 h after which 5 ml samples were acidified by addition of 10 ml 5% acetic acid, and desalted with C18 Zip Tips (see above) prior to MALDI-TOF MS analysis. The remainder of the reaction mixture was subjected to further purification by RP-HPLC on an analytical C18 column using a 0-60% water-ACN gradient containing 0.1% TFA. The identity of putative HNP-containing species was confirmed by MALDI-TOF MS analysis of reduced (DTT) and alkylated (iodoacetamide) samples which produced a new species with the mass of hexa-S-carboxyamidomethylated peptide derivative (+348 amu) [47] .