Chemo-Enzymatic Detection of Protein Isoaspartate Using Protein Isoaspartate Methyltransferase and Hydrazine Trapping

Abstract

Isoaspartate formation is a ubiquitous post-translation modification arising from spontaneous asparagine deamidation or aspartate isomerization. The formation of isoaspartate inserts a methylene group into the protein backbone, generating a "kink", and may drastically alter protein structure and function, thereby playing critical roles in a myriad of biological processes, human diseases, and protein pharmaceutical development. Herein, we report a chemo-enzymatic detection method for the isoaspartate protein, which in particular allows the affinity enrichment of isoaspartate-containing proteins. In the initial step, protein isoaspartate methyltransferase selectively converts isoaspartates into the corresponding methyl esters. Subsequently, the labile methyl ester is trapped by strong nucleophiles in aqueous solutions, such as hydrazines to form hydrazides. The stable hydrazide products can be analyzed by standard proteomic techniques, such as matrix-assisted laser desorption ionization and electrospray ionization mass spectrometry. Furthermore, the chemical trapping step allows us to introduce several tagging strategies for product identification and quantification, such as UV-vis and fluorescence detection through a dansyl derivative. Most significantly, the hydrazide product can be enriched by affinity chromatography using aldehyde resins, thus drastically reducing sample complexity. Our method hence represents the first technique for the affinity enrichment of isoaspartyl proteins and should be amendable to the systematic and comprehensive characterization of isoaspartate, particularly in complex systems.

Discovery of a Chemical Modification by Citric Acid in a Recombinant Monoclonal Antibody

“…One major reason is to minimize the deamidation of asparagine, a spontaneous nonenzymatic process that occurs in all monoclonal antibodies and the vast majority of protein pharmaceuticals. 16,22,24−26,46,47 Specifically, mildly acidic pH has been shown to reduce deamidation of asparagine. 22−27 …”

Mass Spectrometry Based Mechanistic Insights into Formation of Tris Conjugates: Implications on Protein Biopharmaceutics

“…The nucleophilic functional group with free amine in the chemical molecule like hydrazines and hydroxylamines can potentially react with the cyclic succinimide intermediate formed as part of degradation pathway in the asparagine residue with all the possible factors leading to a covalent adduct [35,[38][39][40][41][42][43][44][45][46]. Tris with its free amine, a potential nucleophilic functional group, reacts with the cyclic asparagine-succinimide intermediate leading to the formation of in-solution covalent adduct that gives the mass of 104 Da addition at the asparagine site adjacent to the glycine residue (Scheme 1).…”

Mildly acidic conditions eliminate deamidation artifact during proteolysis: digestion with endoprotease Glu-C at pH 4.5

“…As isoAsp has detrimental impacts on biological systems, several repair mechanisms are present for reducing the levels of isoAsp; one prominent pathway is through protein isoaspartate O-methyltransferase (PIMT or PCMT, EC 2.1.1.77). This enzyme catalyzes the transfer of a methyl group from S-adenosyl-L-methionine (AdoMet or SAM) to isoAsp, generating an isoaspartyl methyl ester that can rapidly hydrolyze back to Asp (Alfaro et al 2008; Biastoff et al 2006; Mosley et al 2006; Zang et al 2009; Zhou et al 1999). The repair of isoAsp via PIMT methylation is also affected by metabolites in the one-carbon and transsulfuration pathways, such as homocysteine (Gui et al 2013; Mosley et al 2006; Perła-Kaján and Jakubowski 2012; Perła-Kaján et al 2007).…”