Revealing deamidation and isoaspartate formation during peptide analysis, purification and storage by tandem mass spectrometry.

Interest in peptides and peptidomimetics continues to grow, particularly in the context of drug discovery and development. However, spontaneous chemical modifications such as deamidation and isoaspartate formation present significant challenges, as they are difficult to detect and can compromise peptide integrity and function. Conventional chromatographic methods and standard mass spectrometric analyses often fail to distinguish structurally similar peptides with nearly identical physicochemical properties and masses. In this study, tandem mass spectrometry (MS/MS) was employed to monitor deamidation events involving asparagine, glutamine and C-terminal amide functional groups. Both collision-induced dissociation (CID) and electron-transfer dissociation (ETD) were systematically evaluated and could differentiate the resulting species without relying on chromatographic separation, with ETD further enabling semi-quantitative detection of deamidation and isoaspartate formation. Using this approach, we confirmed isoaspartate formation under mildly basic conditions such as phosphate-buffered saline, whereas amidated peptides remained stable in neutral aqueous-organic mixtures or at lower temperatures. In contrast, exposure to acidic conditions, particularly in the presence of the additive trifluoroacetic acid, as commonly used during HPLC purification, resulted in substantial direct deamidation by hydrolysis without detectable isoaspartate formation. Notably, this degradation showed clear site dependence, with especially C-terminal amides, being markedly more susceptible in our study. These findings underscore how readily deamidation and isoaspartate formation can occur under routine laboratory conditions and highlight the utility of CID and ETD mass spectrometry for reliably detecting these modifications. The study emphasizes the need for careful analytical monitoring during peptide synthesis and purification to avoid misinterpretation of structural integrity.