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

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.

Dissociation Pattern of Sodiated Amide Peptides as a Tool for De Novo Sequencing.

As part of the de novo sequencing issue, new approaches have to be found to sequence small natural peptides (<15-20 residues), which often present amino acid compositions, inducing merely singly charged species, that are quite difficult to thoroughly fragment under low-energy activation conditions in MS/MS experiments. Cationization by alkali metals, like Na+, followed by collision-induced dissociations (CID) or the postsource metastable decay (PSD) of such cationized molecular ions was found to improve the sequence coverage of native peptides through the formation of [bn-1 + Na + OH]+ ions issued from C-terminal residue exclusion. Concerned by the identification of peptides with a C-terminal amide, the fragmentation pattern of their sodiated molecular ions was investigated. In contrast to the peptides featuring unmodified C-termini, the C-terminal loss did not occur, with the amide function triggering different fragmentation pathways. However, several residues, such as aspartic acid (D), glutamic acid (E), and arginine (R), influenced the dissociation of fixed-charge sodiated ions similarly to protonated peptides; more surprisingly, serine (S), threonine (T), and tyrosine (Y), which exhibit a hydroxyl function on their side chains, showed a very peculiar behavior that could help de novo peptide sequencing.