Deamidation as a widespread phenomenon in two-dimensional polyacrylamide gel electrophoresis of human blood plasma proteins

The human plasma protein patterns obtained by two-dimensional polyacrylamid e gel electrophoresis (2-D PAGE) is a good model system for post-translatio nal modifications because of the existence of several "ladders" of protein spots [Anderson, N. L., Anderson, N. G., Electrophoresis 1991, 12, 883-906] , so-called "trains" of spots. Our investigation of several proteins, among st others beta(2)-microglobulin and the haptoglobin chains, found the diffe rences in isoelectric points (pl) to be due to deamidation of asparagines. After enzymatic cleavage with endopeptidases in the 2-D polyacrylamide gel, the asparagine and deamidated asparagine containing peptides were separate d and quantified by reversed-phase HPLC. In order to separate these peptide s, a neutral pH system was established and, as a result, the differences in hydrophobicity of asparagine-containing and deamidated asparagine-containi ng peptides increased. But how do deamidated asparagines contribute to the observed spot pattern? One spot in the 2-D gel consists of a mixture of pro tein species with the same number of deamidated asparagines but on differen t sequence position sites. The difference between the spots in the "ladder" is a growing number of negative charges introduced in the protein by an in creasing number of deamidated asparagines. As a consequence, the mass diffe rence between two spots is exactly 1 Da, which is shown in this paper for i ntact protein masses and the corresponding deamidated peptides.