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 polyacrylamide gel electrophoresis (2‐D PAGE) is a good model system for post‐translational 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, amongst others beta2‐microglobulin and the haptoglobin chains, found the differences in isoelectric points (pI) 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 separated and quantified by reversed‐phase HPLC. In order to separate these peptides, a neutral pH system was established and, as a result, the differences in hydrophobicity of asparagine‐containing and deamidated asparagine‐containing 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 protein species with the same number of deamidated asparagines but on different sequence position sites. The difference between the spots in the „ladder”︁ is a growing number of negative charges introduced in the protein by an increasing number of deamidated asparagines. As a consequence, the mass difference between two spots is exactly 1 Da, which is shown in this paper for intact protein masses and the corresponding deamidated peptides.

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