Protein Identification by Peptide Mass Fingerprinting using MALDI-TOF Mass Spectrometry

Abstract

Developments in mass spectrometry technology, together with the availability of extensive DNA and protein sequence databases and software tools for data mining, has made possible rapid and sensitive mass spectrometry-based procedures for protein identification. Two basic types of mass spectrometers are commonly used for this purpose; MALDI-TOF-MS and ESI-MS. MALDI-TOF instruments are now quite common in biochemistry laboratories and are very simple to use, requiring no special training. ESI instruments, usually coupled to capillary/nanoLC systems, are more complex and require expert operators. We will therefore focus on the use of MALDI-TOF-MS, although the sample preparation is identical for both methods. The principle behind the use of MALDI-TOF-MS for protein identification is that the digestion of a protein with a specific protease will generate a mixture of peptides unique to that protein. Measuring the molecular masses of these peptides then gives a characteristic dataset called a peptide mass fingerprint (PMF) (1). The PMF data can then be compared with theoretical peptide molecular masses that would be generated by using the same protease to digest each protein in the sequence database, to find the best match. Provided the protein being analyzed is present in the database being searched and the data is of sufficient quality, the best match should be the correct protein. In order to judge the validity of a protein identification by this method, some means of scoring the quality of the match must be used. The procedure described here involves cutting protein bands or spots from 1-D or 2-D PAGE gels, destaining the gel pieces, reducing and alkylating the