Gas-Phase Peptide Fragmentation Induced by Hydrogen Attachment, from Principle to Sequencing of Amide Nitrogen-Methylated Peptides.

Tandem mass spectrometry (MS/MS) with radical-based fragmentation was developed recently, which involves the reaction of hydrogen atoms and peptides in a process called hydrogen attachment/abstraction dissociation (HAD). HAD mainly produces [c + 2H] and [z + 2H] via hydrogen attachment to the carbonyl oxygen on the peptide backbone. In addition, HAD often generates [a + 2H] and [x + 2H]. To explain the formation of [a + 2H] and [x + 2H], hydrogen attachment to the carbonyl carbon atom on the peptide backbone is proposed to initiate C-C bond cleavage. The resultant hydrogen-abundant oxygen-centered radical intermediate undergoes radical-induced dissociation to give [a + H] and [x + 2H]. Subsequently, [a + 2H] was produced by the reaction of [a + H] and a hydrogen atom. The fragment ions formed by the cleavage of N-C and C-C bonds are observed in the HAD-MS/MS spectra, and the mass differences of these fragment ions correspond to the mass of peptide bonds. Consequently, HAD-MS/MS allows the identification of post-translational modifications on the peptide backbone. In addition, HAD-MS/MS provides a consecutive series of [c + 2H] and [a + 2H] as the N-terminal fragments, as well as [z + 2H] and [x + 2H], which enables the sequencing of peptides with post-translational modification, including the discrimination of modifications on the side chain and backbone.