Chemical Methods for the Detection of Protein N-Homocysteinylation via Selective Reactions with Aldehydes

Abstract

Elevated blood levels of homocysteine (Hcy), hyperhomocysteinemia or homocystinuria, have been associated with various diseases and conditions. Homocysteine thiolactone (Hcy TL) is a metabolite of Hcy and reacts with amine groups in proteins to form stable amides, homocystamides or N-homocysteinylated proteins. It has been proposed that protein N-homocysteinylation contributes to the cytotoxicity of elevated Hcy. Due to its heterogeneity and relatively low abundance, detection of this post-translational modification remains challenging. On the other hand, the gamma-aminothiol group in homocystamides imparts different chemical reactivities than the native proteins. Under mildly acidic conditions, gamma-aminothiols irreversibly and stoichiometrically react with aldehydes to form stable 1,3-thiazines, whereas the reversible Schiff base formation between aldehydes and amino groups in native proteins is markedly disfavored due to protonation of amines. As such, we have developed highly selective chemical methods to derivatize N-homocysteinylated proteins with various aldehyde tags, thereby facilitating the subsequent analyses. For instance, fluorescent or biotin tagging coupled with gel electrophoresis permits quantification and global profiling of complex biological samples, such as hemoglobin and plasma from rat, mouse and human; affinity enrichment with aldehyde resins drastically reduces sample complexity. In addition, different reactivities of lysine residues in hemoglobin towards Hcy TL were observed.

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).…”

The application of a chemical determination of N-homocysteinylation levels in developing mouse embryos: implication for folate responsive birth defects

“…Herein we present the application of a novel technique of the chemical determination of N-homocysteinylation[41] that has been utilized to address the hypothesis that N-homocysteinylation can contribute to NTD susceptibility. The assay has been performed in murine embryos collected directly following the period of neural tube closure.…”

“…The chemical determination for N-homocysteinylation described by Zang, et al[41], was modified here to interrogate a large set of biological samples. This new approach represents an easily-accessible and highly specific assay for a stoichiometric quantification of N-homocysteinylation events.…”

Discovery and elucidation of unknown protein modifications : brominated coumarin azide as a probe for alkynes

“…We foresee that this tag and workflow will be particularly powerful for more complex systems including detecting protein modifications and drug targets in the cellular milieu [44][45] and systems with undefined chemistry and/or sites for bioconjugation or degradation, and moreover, novel chemistry 46 and biology.…”

“…We will discuss several examples of how scientists first discovered unknown modifications using various analytical techniques and then used various mass spectrometry methodologies to elucidate the novel chemistry, mechanism and sites of the protein modification, to ultimately understand the thought process of the workflows for discovery and elucidation. [45][46][47] In addition to the main peak, there are many peaks appearing in the early eluting (acidic) and late eluting (basic) regions of the chromatogram. For therapeutic development, it is essential to understand the causing factors, chemistry and mechanism of the protein modifications that occur to develop a control strategy that minimizes the impact on product quality.…”