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Introduction: Microwave-assisted acid hydrolysis has recently been reported in the literature as a tool for bottom-up proteomic analysis as well as increase coverage of proteins when traditional proteolytic cleavage sites are not present, and also for identification and characterization of the termini of proteins. Here two microwave-assisted acid proteolysis methods were compared, one involved the use of trifluoroacetic acid (TFA) and the other with formic acid. In this experiment, we monitored the; acid cleavage specificity; reaction / preparation time overall protein coverage; and N- and C-terminal coverage.
Materials and Methods: Standard proteins (1–5 μg) were diluted into two acids 1–5 M TFA and 1%–50% formic acid, which were subjected to microwave irradiation using an industrial microwave system (CEM Corp., Matthew, NC). Samples were microwave irradiated for 1–5 min and resulting peptide fragments were then subjected to reverse-phase separation, followed by MSn analysis on an LCQ-Deca Ion Trap mass spectrometer (Thermo Electron, San Jose, CA) fragmenting the three most intense peaks. MS/MS data were searched with Mascot (Matrix, London) for protein coverage and identification with ‘no enzyme’ selected in the search parameters.
Results: Preliminary results show that controllable cleavage at specific proteolytic sites can be obtained when employing set acid concentrations under controlled microwave irradiation. As previously reported, formic acid can cleave preferentially at Aspartic acid. TFA can be manipulated to cleave at either preferentially the N and occasionally C-terminus of a protein, with longer incubation times leading to internal cleavage at acid labile bonds.
Conclusion: Microwave-assisted acid proteolysis can be used either as an alternative proteolytic reagent with less sequence constraints than traditional enzymes. Conditions can be manipulated to direct proteolysis to mainly the termini of the protein, or indeed internal cleavage sites.