Protein Identification by Peptide Mass Fingerprinting

Abstract

This chapter describes the database identification of proteins that have been separated by one-(or two-) dimensional gel electrophoresis followed by in-gel digestion of the protein bands or spots from the gels. Database searches using mass spectrometry data are particularly important to identify a specific protein and to determine whether post-translational modifications are present. Use of some of the many websites for “eomic”identification of proteins is described here. Various software, including commercial software packages such as SEQUEST (see Note 1), is available to use the information on the fragment ions obtained from a tandem MS experiment to search protein (and DNA translation) databases to identify the sequence and the protein from which it is derived. The increasing number and public availability of complete genome sequences has greatly increased the possibility of protein identification using intact peptide mass data (fingerprints). Fingerprints of digests of complex protein mixtures are unsuitable, since it would not be possible to ascertain which peptide(s) originated from which protein component in such a mixture. Matrix-assisted laser desorption/ ionization (MALDI)-time-of-flight (TOF) mass spectrometry (MS) has a particular application here, due to the possibility of high throughput with the assistance of robotics to excise large numbers of spots from 2-D gels, digest, and spot onto a MALDI plate. Modern MALDI-TOF MS instruments now have the sufficiently high mass accuracy necessary for unambiguous identification based on fingerprint data (1).