Direct N-terminal sequencing of polypeptides using a thermostable bacterial aminopeptidase and MALDI-TOF mass spectrometry

Mass spectrometry-based amino acid sequencing is currently based almost entirely on collision-induced peptide fragmentation and analyses. Here, we describe a single-stage MS-based technique for amino acid sequencing involving partial, heterogenous digestion of a peptide by a processive, non-specific, heat-loving Bacillus subtilis-derived aminopeptidase (BsuAP), which acts optimally at 70 °C and allows ‘single-shot’ sequencing to be carried out through simultaneous accumulation, and detection of sub-populations of peptides of progressively reducing length.