Capillary electrophoresis-tandem mass spectrometry as a highly selective tool for the compositional and site-specific assessment of multiple peptide-deamidation

•A new approach to assess composition of multiple deamidated peptides is described.•Separation of multiple deamidated (including isobaric) species is achieved by CE.•Tandem-MS allows determination of deamidation sites.•Peptide composition is determined using isotopic-like distributions of fragment ions.•Applicability is proved for samples exposed to different stress conditions.

Site-specific mapping of multiple deamidations in peptides is a challenging analytical task. In this work, capillary electrophoresis-tandem mass spectrometry (CE-MS/MS) is presented as a high-resolution tool for the detailed characterization of these subtle modifications in peptides. The 4.5-kDa peptide drug TRI-1144, which contains five closely-positioned potential deamidation sites, was selected as model compound. TRI-1144 was exposed to acidic conditions and/or elevated temperatures for 1-14 h. Stressed samples were analyzed using a background electrolyte (BGE) of 150 mM ammonium formate (pH 6.0) in combination with a capillary coated with a bilayer of Polybrene-dextran sulfate. Separation of deamidated and deacetylated TRI-1144 species, including several positional isomers, was greatly enhanced by adding up to 40 vol% of acetonitrile-isopropanol (87.5:12.5, v/v) to the BGE, allowing reliable determination of the number of deamidations/deacetylations per degradation product. Collision-induced dissociation MS/MS was conducted on the separated peptide components in order to reveal the exact position of deamidation on the peptide chain. Obtained fragment ions showed overlapping isotopic distributions in their MS/MS spectra resulting from the comigration of different isomeric deamidated species. Comparison of theoretical and measured isotope distributions for specific y ions of peptide fragments yielded the identity and relative abundance of isomeric deamidated products. The developed CE-MS/MS methodology was used for the highly selective evaluation of TRI-1144 stability under different stress conditions, providing detailed qualitative and semi-quantitative degradation maps of the peptide drug.

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