We have developed a method for aminopeptidase-based sequence determination of peptides by MS (e.g., ordinary MALDI-TOF mass spectrometry) instead of by MS-MS mass spectrometry. Briefly, the TET aminopeptidase from Bacillus subtilis (which is a processive aminopeptidase whose mechanism of action we have been otherwise studying) is reacted with a peptide at 70 degrees (the optimum temperature of activity of the enzyme) for a definite length of time. Different populations of the peptide are cleaved at their N-termini to different extents by the processive aminopeptidase. This leads to the accumulation of peptides of various (all possible) lengths, in the same reaction, allowing all masses to be simultaneously determined through a MALDI-TOF experiment. The differences in the masses of peptides of different lengths which are generated equal the masses of different individual amino acids, and this provides information about the sequence of the peptide. The demonstration of this technique constitutes the first practical demonstration of the use of an aminopeptidase to perform peptide sequencing through simple MS mass spectrometry. Combined with independent LC-based separation of tryptic fragments of any protein, this technique can allow any laboratory with a simple MALDI-TOF-based mass spectrometer to sequence large sections of any protein, by combining trypsin-based peptide generation with chymotrypsin-based and V8 protease-based peptide generation.

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