Abstract
Protease mapping is an established method for probing the primary structure of proteins (1,2) and has traditionally been performed through the use of chromatography and/or gel electrophoresis techniques in combination with Edman degradation NH2-terminal sequencing (3). More recently, mass spectrometry has been combined with protease mapping to perform “protein mass mapping.” Definitively, protein mass mapping combines enzymatic digestion, mass spectrometry, and computer-facilitated data analysis to examine proteolytic fragments for protein structure determination. Protein mass mapping permits the identification of protein primary structure by applying sequence specific proteases and performing mass analysis on the resulting proteolytic fragments, thus yielding information on fragment masses with accuracy approaching ±5 ppm, or ±0.005 Daltons for a 1000 Daltons peptide. The protease fragmentation pattern is then compared with the patterns predicted for all proteins within a database, and matches are statistically evaluated. Since the occurrence of Arg and Lys residues in proteins is statistically high, trypsin cleavage (specific for Arg and Lys) generally produces a large number of fragments, which, in turn, offer a reasonable probability for unambiguously identifying the target protein. The success of this strategy relies on the existence of the protein sequence within the database, but with the sequences of whole genomes for several organisms now complete (Escherichia coli, Bacillus subtilis, and Archaeoglobus fulgidus) and others approaching completion (Saccharomyces cerevisiae, Saccaramyces pombe, Homo sapiens, Drosophila melanogaster, and so forth), the likelihood for matches is reasonably high.
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W. Kriwacki, R., Siuzdak, G. (2000). Probing Protein-Protein Interactions with Mass Spectrometry. In: Chapman, J.R. (eds) Mass Spectrometry of Proteins and Peptides. Methods in Molecular Biology™, vol 146. Humana Press, Totowa, NJ. https://doi.org/10.1385/1-59259-045-4:223
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DOI: https://doi.org/10.1385/1-59259-045-4:223
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