Strategies for Protein Identification

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Protein identification may be done by various methods:

Peptide mass fingerprinting

We have studied two-dimensional gel electrophoresis in earlier lectures. We have also studied how over or under expression of a protein spot is identified. Once protein spot of interest is identified, the same is excised and digested by specific protease such as trypsin. As trypsin is very specific in the cleavage each protein sequence will result in specific set of peptides of varying masses that are characteristic of that protein. The mass of each peptide will be the sum of the amino acids present including any modifications that those amino acids might have undergone. The masses of these peptides are measured by mass spectrometry and then in silico compared to either a database containing known protein sequences or even the genome. Computer programs translate the known genome of the organism into proteins, then theoretically cut the proteins into peptides with the same protease (for example trypsin), and calculate the absolute masses of the peptides from each protein. They then compare the masses of the peptides of the unknown protein to the theoretical peptide masses of each protein encoded in the genome. The results are statistically analyzed to find the best match (Fig. 1). Some additional details about the protein, if known, like molecular mass, modifications etc may further help in protein identification.