What is the Structure of b2 Ions Generated from Doubly Protonated Tryptic Peptides?

A recent statistical study (Savitski, M. M.; Falth, M.; Eva Fung, Y. M.; Adams, C. M.; Zubarev, R. A. J. Am. Soc. for Mass Spectrom. doi: 10.1016/j.jasms.2008.08.003) of a large spectral database indicated that the product ion spectra of doubly protonated tryptic peptides fall into two distinct classes. The main factor distinguishing the two classes is the relative abundance of the y(N-2) fragment: for Class I spectra y(N-2) is the most abundant y fragment while for Class II other y ions dominate the corresponding spectra. To explain the dominance Of y(N-2) for Class I spectra formation of a nontraditional b(2) ion with a diketopiperazine (6-membered cyclic peptide) rather than an oxazolone structure was proposed. Here we present evidence from tandem mass spectrometry, hydrogen/deuterium exchange, and density functional calculations that do not support this proposal. Namely, that CID of doubly protonated YIGSR, YGGFLR, and YIYGSFK produce Class I product ion spectra, yet the b2 fragment is shown to have the traditional oxazolone structure. (J Am Soc Mass Spectrom 2009, 20, 618-624) (C) 2009 Published by Elsevier Inc. on behalf of American Society for Mass Spectrometry