Deamidation and succinimide formation by gamma-N-methylasparagine: potential pitfalls of amino acid analysis.

The biological function of the post-translationally methylated amino acid gamma-N-methylasparagine (gamma-NMA) in proteins is unknown. We are examining the premise that amide methylation protects against deamidation. The free amino acids Asn, gamma-NMA, Gln, and delta-N-methylglutamine (delta-NMG) were incubated at elevated temperature and a variety of pH conditions to assay for deamidation. Gln disappears 12- to 14-fold more rapidly than delta-NMG, and Asn hydrolyzes to Asp and NH3 as expected. However, the gamma-NMA deamidation rate is severely overestimated by simply measuring the disappearance of starting material because gamma-NMA undergoes a cyclization reaction in preference to deamidation. At pH 1 the predominant gamma-NMA reaction is formation of stable 3-amino-N-methylsuccinimide (NMS) and this occurs greater than 10-fold faster than Asn deamidation. At pH 4.0, 7.4, and 9.0 NMS is readily formed but it is unstable and partitions between the parent compound, gamma-NMA, and a second species, alpha-N-methylasparagine. At pH 7.4 and 9.0 gamma-NMA disappears 4-fold slower than Asn but the methyl amide hydrolysis rate is diminished by as much as 13-fold. The Asn incubations over the pH range 1-9 yield scant evidence of a succinimide intermediate. It is concluded that the amide methylation provides a unique reaction pathway and stabilization for the N-methylsuccinimide species. Amino acid analysis by o-phthalaldehyde postcolumn reaction fails to detect isoasparagine, alpha-N-methylasparagine, and NMS. Amino acid analysis by precolumn derivatization with phenyl isothiocyanate destroys NMS and therefore cannot quantitate this compound. The ninhydrin postcolumn derivatization method is able to detect and quantitate all of these amino acid species.