Peptide Mass Mapping of Purified Proteins

The next stage in the analysis includes a proteolytic cleavage of the protein to generate peptides that are amenable to MALDl-MS analysis. MALDI-MS is conveniently used for the initial analysis of the crude peptide mixture in order to read out the generated peptide fragments and for analysis of individual peptides after their separation and isolation by LC. 

Traditionally, proteins were initially characterized by de-novo sequencing using automated Edman degradation and amino acid composition analysis. Today, however, these techniques tend to be replaced by MS, which not only provides more flexibility and sensitivity but is also amenable to the analysis of protein and peptide mixtures. Tandem mass spectrometry (MS/MS) is used for amino acid sequencing of peptides. MALDI-MS/MS is very powerful for peptide characterization and identiflcation via sequencing and sequence database searching. 

Because of this sequestration of the proton to the basic sites in the peptides, MALDI often gives rise to poor MS/MS fragmentation efficiency of peptide ions. Therefore, the applicability of MALDl-MS/MS for the de-novo sequencing of peptides was initially limited. This kindled interest in the development of alternative 

An alternative is to use a metalloendopeptidase with Lys-N cleavage specificity combined with strong cation exchange enrichment. MALDl MS/MS analysis of such peptide ions containing a single lysine residue (and no other basic residue) has been found to result in spectra containing almost exclusively b-ions [37]. 

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