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

An investigation into the applicability of a sequential digestion procedure involving endo- and exoprotease digestion of proteins is reported. The pro cedure involves the digestion of a protein sample with trypsin, yielding pe ptide fragments characteristic of the protein. The resulting mixture of pep tide fragments is then subjected to N-terminal sequencing with leucine amin opeptidase M (LAP), with matrix-assisted laser desorption/ionization time-o f-flight mass spectrometric analysis of the various digestion products. Sev eral proteins in solution, as well as gel-extracted proteins, were subjecte d to this sequential enzyme digestion procedure. The results of these exper iments 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 ge nerated with this method is limited; the longest sequence tag generated fro m a single tryptic peptide was four amino acids, even though the digestion was allowed to proceed for long times. In the experiments, N-terminal diges tion products were detected as early as two minutes, or as late as 90 minut es, following the addition of LAP to the sample. The method was shown to be effective for sub-picomole starting quantities of protein, although with s ome loss in digestion efficiency at lower concentrations of starting materi al. This method is useful in providing additional sequence information to i ncrease the level of confidence in protein identification, as illustrated i n the identification of bacterial proteins fractionated by HPLC. In some in stances, this method can provide additional sequence information where post -source decay and nanospray mass spectrometry failed to generate fragment-i on spectra. This is illustrated by an example where the procedure was appli ed to a membrane protein, CD9, that had been isolated by sodium dodecyl sul fate-polyacrylamide gel electrophoresis. Although the sequential digestion procedure requires more human intervention, it is a straightforward method and can be readily implemented.