Diagnosing the protonation site of b 2 peptide fragment ions using IRMPD in the X-H (X = O, N, and C) stretching region

Charge-directed fragmentation has been shown to be the prevalent dissociation step for protonated peptides under the low-energy activation (eV) regime. Thus, the determination of the ion structure and, in particular, the characterization of the protonation site(s) of peptides and their fragments is a key approach to substantiate and refine peptide fragmentation mechanisms. Here we report on the characterization of the protonation site of oxazolone b ₂ ions formed in collision-induced dissociation (CID) of the doubly protonated tryptic model-peptide YIGSR. In support of earlier work, here we provide complementary IR spectra in the 2800-3800 cm ^-1 range acquired on a table-top laser system. Combining this tunable laser with a high power CO ₂ laser to improve spectroscopic sensitivity, well resolved bands are observed, with an excellent correspondence to the IR absorption bands of the ring-protonated oxazolone isomer as predicted by quantum chemical calculations. In particular, it is shown that a band at 3445 cm ^-1, corresponding to the asymmetric N-H stretch of the (nonprotonated) N-terminal NH ₂ group, is a distinct vibrational signature of the ring-protonated oxazolone structure.