Sequence databases, peptide sequencing using

E Peptide Sequencing Using Mass Spectrometry and Sequence Databases... 

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. 

Figure 10.3 An example of peptide identification with MS/MS. For a peptide of interest at m/z 1 777.97, an MS/MS spectrum is acquired at the precursor mass using the biological sample with the highest intensity level for the peptide. The MS/MS spectrum is searched against a database of human protein sequences using the MASCOT search...

An analogous approach using databases is to infer the DNA sequence that codes for a partial peptide sequence and compare this DNA sequence with the database of known DNA sequences. If a satisfactory match is found, the remaining sequence of the polypeptide can be read from the DNA sequence using the genetic code (see Section 25.5). In addition, the... 

Computer algorithms facilitate identification of the open reading frames that encode a given protein by using partial sequences and peptide mass profiling to search sequence databases. 

The major advantage of the tandem mass spectrometry approach compared to MALDI peptide fingerprinting, is that the sequence information obtained from the peptides is more specific for the identification of a protein than simply determining the mass of the peptides. This permits a search of expressed sequence tag nucleotide databases to discover new human genes based upon identification of the protein. This is a useful approach because, by definition, the genes identified actually express a protein. 

Figure 5.11. Generic approaches to identify interacting proteins within complexes. The complex is isolated from cells by affinity purification using a tag sequence attached to a protein known to be in the complex. Alternatively, the complex can be immunprecipitated with an antibody to one of the proteins in the complex. The proteins are resolved by polyacrylamide gel electrophoresis, proteolyzed, and the mass of the resulting peptides is determined by mass spectrometry. Alternatively, the proteins can be proteolyzed and the resulting peptides resolved by liquid chromatography. The peptide masses are then determined by mass spectrometry and used for database searching to identify the component proteins.

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