Tryptic digestion and protein identification for protease inhibition studies

Tryptic peptides derived from each gel spot were submitted for identification by PMF using MALDI-TOF MS. Dried samples were reconstituted in 0.1% TFA (typically 10-20 mL), and mixed 1:1 with matrix solution (a-cyano-4-hydroxy-cinnamic acid, 5 or 10 mg/mL in 70% ACN, with 0.03% TFA). Aliquots (1 mL) were spotted on the MALDI target and allowed to dry at room temperature before introduction into the mass spectrometer. Mass spectra were acquired in positive ion reflection mode after close-external calibration using bradykinin 1-7 and insulin oxidized B-chain as standards. Spectra were typically summed over 100 shots. A Shimadzu-Biotech Axima CFRâ„¢plus instrument was employed for this work. Protein identification was performed using MASCOT database searching at http://www.matrixscience.com. Search parameters were restricted to Homo sapiens taxonomy for use with the NCBInr database. Enzyme selection was trypsin, with up to one missed cleavage permitted. Carbamidomethylation of cysteines was selected as a fixed modification. Protein mass was unrestricted, and peptide mass tolerance typically set at 250 ppm. Mass values were entered as monoisotopic MH + . Mass lists were generated automatically using the MASCOT Wizard software application, with a S/N threshold of 100.

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