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Article Dans Une Revue Talanta Année : 2022

Quantitating α-amidated peptide degradation by separative technologies and ultra-high resolution mass spectrometry

Résumé

α-amidation of peptides is a C-terminal modification allowing to improve half-life of therapeutic peptides. However, due to storage conditions, this C-terminal amide function can be partially hydrolyzed into a carboxylic acid moiety, resulting in a decrease of the molecule bioactivity. It is therefore necessary to be able to detect and quantify any trace of this degradation product when dealing with such class of peptides. For this purpose, LC-UV, CE-UV, LC/MS and LC/MS/MS are conventional analytical technologies used in pharmaceutical laboratories to monitor peptide integrity. Since the production of the carboxylated peptide issued from the amidated starting compound is scrutinized, ultra-high-resolution mass spectrometry was investigated without the recourse to any separative method (direct infusion mode) to achieve rapid straightforward relative quantitative analyses from the detected isotopic clusters of both peptides. Indeed, low or high-resolution MS instruments present in phar- maceutical laboratories (fitted with Q, IT or ToF mass analyzers) allow to distinguish both molecular ions that differ from 0.984 Da (NH2 formally substituted by OH). However, the analytical difficulty comes from the fact that the A+1 ion (13C contribution) of the amidated peptide and the A ion (12C contribution) of any produced carboxylated sequence are overlapping hampering the detection of minute amount of the hydrolyzed product. Actually, to differentiate these two isobaric ions, mass analyzers should possess at least a resolution of 90 000 for 7 residues peptides (molecular ions around 700–800 Da) which is presumably attainable with any FT-MS in- strument. With an ultra-high-resolution mass spectrometer (FT-ICR MS) the observed fine isotopic structure displaying all expected isotopologues permitted in a single fast analysis to unambiguously detect both com- pounds (non-hydrolyzed and hydrolyzed one) and subsequently define the relative amount of the carboxylated peptide contaminant. Finally, these results were confronted with conventional measurements recorded with orthogonal methodologies such as LC-UV and CE-UV. The pro and cons of all quantitative strategies are discussed.
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Dates et versions

hal-04016606 , version 1 (06-03-2023)

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Elodie Logerot, Catherine Perrin, Yoann Ladner, Frédéric Aubriet, Vincent Carré, et al.. Quantitating α-amidated peptide degradation by separative technologies and ultra-high resolution mass spectrometry. Talanta, 2022, 253, pp.124036. ⟨10.1016/j.talanta.2022.124036⟩. ⟨hal-04016606⟩
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