H. Sarioglu et al., Deamidation as a widespread phenomenon in two-dimensional polyacrylamide gel electrophoresis of human blood plasma proteins, ELECTROPHOR, 21(11), 2000, pp. 2209-2218
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.