Identification of Proteins by Amino Acid Composition After Acid Hydrolysis

Abstract

In a proteome project, hundreds or even thousands of spots are produced on a single two-dimensional (2-D) gel or blot, each spot being a different protein or an isoform. Identification of these proteins from the few picomoles present in the spot presents a challenge to protein chemists. One technique for identification available in many laboratories and which does not incur great cost is amino acid composition. Latter et al. (1984) first described identification of 2-D separated proteins with their amino acid compositions using radiolabelling of amino acids. They determined the composition by radiolabelling proteins with one radioactive amino acid at a time, calculating the ratios of amino acids by quantitative optical density measurements from different gels, and comparing these ratios against databases. This approach is described in Chapter 8 (this vol.) by Labarre and Perot. An alternative approach is to use acid hydrolysis of proteins followed by quantitation of the resulting free amino acids by chromatography, and the matching of data against databases (Eckerskorn et al. 1988; Jungblut et al. 1992; Shaw, 1993; Hobohm et al. 1994; Galat et al. 1995; Wilkins et al. 1996a). The matching procedure is aided by computer programs, some that are now accessible via the internet, which use amino acid composition together with pI and molecular weight estimates to produce lists of likely protein identifications. Some programs also allow for the inclusion of an N-terminal sequence tag and species.

This Chapter is dedicatd to the memory of Yik Fung.