Investigation of the applicability of a sequential digestion protocol using trypsin and leucine aminopeptidase M for protein identification by matrix‐assisted laser desorption/ionization – time of flight mass spectrometry

An investigation of the applicability of a sequential digestion procedure involving endo‐ and exoprotease digestion of proteins is reported. The procedure involves the digestion of a protein sample with trypsin, yielding peptide fragments characteristic of the protein. The resulting mixture of peptide fragments is then subjected to N‐terminal sequencing with leucine aminopeptidase M (LAP), with matrix‐assisted laser desorption ionization‐time of flight mass spectrometric analysis of the various digestion products. Several proteins in solution, as well as gel extracted proteins were subjected to this sequential enzyme digestion procedure. The results of these experiments reveal that LAP will preferentially cleave specific peptides of the trypsin digested sample with high efficiency, while leaving other peptides undigested. Also, the length of the amino acid sequence tags that can be generated with this method is limited; the longest sequence tag generated from a single tryptic peptide was four amino acids, even though the digestion was allowed to proceed for long times. In the experiments, N‐terminal digestion products were detected as early as two minutes, or as late as 90 minutes, following the addition of LAP to the sample. The method was shown to be effective for subpicomole starting quantities of protein, although with some loss in digestion efficiency at lower concentrations of starting material. This method is useful in providing additional sequence information to increase the level of confidence in protein identification, as illustrated in the identification of bacterial proteins fractionated by high‐performance liquid chromatography. In some instances, this method can provide additional sequence information where post source decay and nanospray mass spectrometry failed to generate fragment ion spectra. This is illustrated in an example where the procedure was applied to a membrane protein, CD9, that had been isolated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Although the sequential digestion procedure requires more human intervention, it is a straightforward method and can be readily implemented.