Proton affinities of the N- and C-terminal segments arising upon the dissociation of the amide bond in protonated peptides.

Dissociation of the amide bonds in a protonated peptide leads to N-terminal sequence fragments with cyclic structures and C-terminal sequence fragments with linear structures. The ionic fragments containing the N-terminus (bn) have been shown to be protonated oxazolones, whereas those containing the C-terminus (Yn) are protonated linear peptides. The coproduced neutral fragments are cyclic peptides from the N-terminus and linear peptides from the C-terminus. A likely determinant of these structural choices is the proton affinity (PA) of the described peptide segments. This study determines the PA values of such segments (Pep), i.e., cyclic and linear dipeptides and a relevant oxazolone, based on the dissociations of proton-bound dimers [Pep + Bi]H+ in which Bi is a reference base of known PA value (Cooks kinetic method). The dissociations are assessed at different internal energies to thereby obtain both proton affinities as well as entropies of protonation. For species with comparable amino acid composition, the proton affinity (and gas phase basicity) follows the order cyclic peptide << oxazolone approximately linear peptide. This ranking is consistent with dissociation of the protonated peptide via interconverting proton-bound complexes involving N-terminal oxazolone (O) or cyclopeptide (C) segments and C-terminal linear peptide segments (L), viz. O...H+...L reversible C...H+...L. N-terminal sequence ions (bn) are formed with oxazolone structures which can efficiently compete for the proton with the linear segments. On the other hand, N-terminal neutral fragments detach as cyclic peptides, with H+ now being retained by the more basic linear segment from the C-terminus to yield Yn.

Characterization of neutral fragments in tandem mass spectrometry: a unique route to mechanistic and structural information.

The neutral species eliminated upon fragmentation of fast-moving mass-selected ions can be directly identified by collisional ionization and detection in neutral fragment reionization (Nf R) mass spectra. Establishment of the identity of neutral fragments yields valuable insight into the decomposition mechanism of a precursor ion, as demonstrated for fullerene and alkali metal iodide cluster ions as well as metal ion adducts of amino acids. In addition, neutral fragment reionization also provides structural information that may not be available from the complementary ionic fragments alone; this is illustrated in the differentiation of isomeric mononucleotides. The parameters influencing the appearance of Nf R spectra are discussed and the scope and general applicability of the method are briefly evaluated.

Differentiation of N-from C-protonated aniline by neutralization-reionization.

Amino- and ring-protonated aniline are distinguished in the gas phase by neutralization-reionization mass spectrometry. This method takes advantage of the dramatically different stabilities and reactivities of the neutralized forms of N- and C-protonated aniline, to ascertain thereby the specific protonation site(s). Fast atom bombardment ionization of aniline is found to yield primarily the anilinium cation (N-protonated tautomer). In contrast, chemical ionization with a variety of reagent gases is shown to generate mixtures in which the ring-protonated species predominates.