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

Abstract

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 (p/) 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.

Fragmentation of Proteins in Cartilage Treated with Interleukin-1

“…The most abundant fragment detected on two-dimensional gels, with an apparent molecular mass of 28 kDa, represented large parts of the globular NC4 domain. The presence of the 28-kDa fragment in spots on the two-dimensional gel over a large interval of isoelectric points might be due to deamidation of asparagine residues (45). Indeed one of the peptides sequenced appeared to exhibit deamidation (Fig.…”

Mitochondria: A mirror into cellular dysfunction in heart disease

“…White et al however, showed both non-deamidated and deamidated forms, suggesting that the modification was not related to sample processing. In addition, it has also been suggested that deamidation at Asn-Gly and Asn-Ser motifs are biologically relevant [161,162], which corresponds with the observed deamidation to a B-crystalline (Asn 146 -Gly 147 ) [160] and may explain the proteolysis of MLC-2 between Asn 13 and Ser 14 [88]. Whilst it appears that myofilament and cytosolic proteins are deamidated and preferentially degraded with I/R injury, the role of deamidation in the mitochondria during pathology has not yet been determined.…”

Glycan Characterization of PSA 2-DE Subforms from Serum and Seminal Plasma

“…Therefore, it seems likely that factors other than N-glycosylation may be modifying the theoretical pI of PSA (7.26), such as changes in the primary sequence or different posttranslational modifications (PTM). Deamidation of asparagines or glutamines have been suggested as modifications responsible for the spot trains of some serum proteins obtained in a 2-DE gel (Sarioglu et al, 2000). The deamidation process generates negatively charged aspartic and glutamic acid residues, which can modify a protein's pI.…”