Deamidation
Deamidation is a chemical reaction in which an amide functional group in the side chain of the amino acids asparagine or glutamine is removed or converted to another functional group. Typically, asparagine is converted to aspartic acid or isoaspartic acid. Glutamine is converted to glutamic acid or pyroglutamic acid (5-oxoproline). In a protein or peptide, these reactions are important because they may alter its structure, stability or function and may lead to protein degradation. The net chemical change is the addition of a water group and removal of an ammonia group, which corresponds to a +1 (0.98402) Da mass increase. Although deamidation occurs on glutamine, glycosylated asparagine and other amides, these are negligible under typical proteolysis conditions.[1]
In the deamidation of an asparagine residue under physiological conditions, the side chain is attacked by the nitrogen atom of the following peptide group (in black at top right of Figure), forming an asymmetric
The rates of deamidation depend on multiple factors, including the primary sequences and higher-order structures of the proteins, pH, temperature, and components in the solutions. Most potential deamidation sites are stabilized by higher order structure. Asn-Gly (NG),is the most flexible and since it is acidic, it is most prone to deamidation with a half-life around 24 h under physiological conditions (pH 7.4, 37 °C).[3]
As a free amino acid, or as the N-terminal residue of a peptide or protein, glutamine deamidates readily to form pyroglutamic acid (5-oxoproline). The reaction proceeds via nucleophilic attack of the α-amino group on the side-chain amide to form a γ-lactam with the elimination of ammonia from the side-chain.
Analytical method
Protein deamidation has been commonly analyzed by reverse-phase liquid chromatography (RPLC) through peptide mapping. Recently reported novel ERLIC-MS/MS method would enhance the separation of deamidated and non-deamidated peptides with increased identification and quantitation quantification.[4]
Mass spectrometry is commonly used to characterize deamidation states of proteins, including therapeutic monoclonal antibodies.[5] The technique is especially useful for deamidation analysis due to its high sensitivity, speed, and specificity. This allows site-specific deamidation analysis.[6]
A major challenge of using mass spectrometry is the formation of deamidation artifacts during sample preparation. These artifacts significantly skew results because they suggest greater rates of spontaneous deamidation than what is truly observed. This can prove problematic in the case of therapeutic proteins which can be mischaracterized in QC protocols if a large percentage of detected deamidation is due to artifacts. Recent studies indicate that lower pH can reduce the rate of deamidation artifacts.[2]
Kinetics of deamidation
Deamidation reactions have been conjectured to be one of the factors that limit the useful lifetime of proteins.[1]
Deamidation proceeds much more quickly if the susceptible amino acid is followed by a small, flexible residue such as
The endoprotease, Glu-C, has shown specificity to only glutamic acid when in specific pH conditions (4.5 and 8.0) and cleaved the C-terminal side when in a solution with Tris-HCl, bicarbonate, or acetate.
See also
References
- Clarke, S (1987). "Propensity for spontaneous succinimide formation from aspartyl and asparaginyl residues in cellular proteins". International Journal of Peptide and Protein Research. 30 (6): 808–821. PMID 3440704.
- Stephenson, RC; Clarke, S (1989). "Succinimide Formation from Aspartyl and Asparaginyl Peptides as a Model for the Spontaneous Degradation of Proteins". The Journal of Biological Chemistry. 264 (11): 6164–6170. PMID 2703484.
- Robinson NE, Robinson AB. (2004) Molecular Clocks: Deamidation of Asparaginyl and Glutaminyl Residues in Peptides and Proteins. Althouse Press: Cave Junction, Ore. OCLC 56978028