Revealing C-terminal peptide amidation by the use of the survival yield technique

• Published in: Analytical biochemistry Vol. 655; p. 114823
• Main Authors: Logerot, Elodie, Cazals, Guillaume, Memboeuf, Antony, Enjalbal, Christine
• Format: Journal Article
• Language: English
• Published: Elsevier Inc 15.10.2022; Elsevier Masson
• Subjects: Amino Acid Sequence; Biochemistry, Molecular Biology; C-terminal amidation; Chemical Sciences; Chromatography, High Pressure Liquid; Chromatography, Liquid; Energy resolved mass spectrometry; Fragmentation; Life Sciences; Peptide; Peptides; Quantitation; Tandem Mass Spectrometry; Energy resolved mass spectrometry; Quantitation; Peptide; C-terminal amidation; Fragmentation
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/j.ab.2022.114823

α-amidation of peptide sequences is a common post-translational modification in the living world. Since the majority of these C-terminal amidated peptides are bioactive, there is hence a great interest to identify and characterize them from biological matrices and natural extracts. Regarding conventional separative methods dedicated to peptides (such as HPLC or CE), elution protocols must be carefully optimized hampering straightforward LC-MS analysis of complex samples. From a mass spectrometry point of view, they are difficult to pinpoint owing to the only 1 Da mass difference between the post-translational amidated and the corresponding native carboxylated forms producing overlapping isotopic contributions of both molecular ions. To circumvent this analytical difficulty, usage of energy-resolved tandem mass spectrometry experiments and of the survival yield technique was investigated. Pair of peptides were thus dissociated in positive and negative mode according to the survival yield technique, in MS2 and MS3 experiments, in order to separate them giving a reliable MS/MS methodology to detect such post-translationally modified sequence. [Display omitted] •Energy resolved mass spectrometry was applied to detect C-terminal amidated peptides.•Carboxylated and amidated sequences display overlapping isotopic clusters.•Sodium molecular adducts showed different profile of breakdown curves for both forms.•Quantitation was evaluated for various molar ratio of carboxylated/amidated forms.