IDENTIFICATION OF YEAST PROTEINS FROM 2-DIMENSIONAL GELS - WORKING OUT SPOT CROSS-CONTAMINATION

With the complete sequence of the yeast genome now available, efforts by many laboratories are underway to identify each of the spots on two -dimensional (2-D) gels corresponding to the most abundant yeast prote ins. The high mass accuracy now attainable using matrix assisted laser desorption/ionization (MALDI)-mass spectrometry equipped with delayed extraction simplifies the process of identification, such that many s pots can be unambiguously identified in a short period of time merely by using peptide mass fingerprinting and generally available database matching programs. Although it is not always possible to match spots b etween gels run by different laboratories, proteins generally yield th e same abundant proteolytic fragments when tryptic digestions are perf ormed. Databases containing these signature peptides not only simplify the task of reidentifying proteins from different gels, but also make it possible to identify small amounts of cross-contaminating proteins from different spots, as well as common extraneous contaminants such as human keratins. In this paper, we present data on the identificatio n of > 20 previously unreported yeast proteins from 2-D gels. Some nov el proteins were identified from randomly analyzed spots. Focusing on 14 spots in a narrow-pH-range gel, we demonstrate how organizing peak- table data and peptide match-list data into databases enables the iden tification of a larger percentage of the peaks.