Structural Influences on Preferential Oxazolone versus Diketopiperazine b₂⁺ Ion Formation for Histidine Analogue-Containing Peptides

Studies of peptide fragment ion structures are important to aid in the accurate kinetic modeling and prediction of peptide fragmentation pathways for a given sequence. Peptide b2 + ion structures have been of recent interest. While previously studied b2 + ions that contain only aliphatic or simple aromatic residues are oxazolone structures, the HA b2 + ion consists of both oxazolone and diketopiperazine structures. The structures of a series of histidine-analoguecontaining Xxx-Ala b2 + ions were studied by using action infrared multiphoton dissociation (IRMPD) spectroscopy, fragment ion hydrogen−deuterium exchange (HDX), and density functional theory (DFT) calculations to systematically probe the influence of different side chain structural elements on the resulting b2 + ion structures formed. The b2 + ions studied include His−Ala (HA), methylated histidine analogues, including π-methyl-HA and τ-methyl-HA, pyridylalanine (pa) analogues, including 2-(pa)A, 3-(pa)A, and 4-(pa)A, and linear analogues, including diaminobutanoic acid−Ala (DabA) and Lys−Ala (KA). The location and accessibility of the histidine π-nitrogen, or an amino nitrogen on an aliphatic side chain, were seen to be essential for diketopiperazine formation in addition to the more typical oxazolone structure formation, while blocking or removal of the τ-nitrogen did not change the b2 + ion structures formed. Linear histidine analogues, DabA and KA, formed only diketopiperazine structures, suggesting that a steric interaction in the HisAla case may interfere with the complete trans−cis isomerization of the first amide bond that is necessary for diketopiperazine formation. ■ INTRODUCTION Elucidation of structural influences on chemical reactions, including unimolecular dissociation reactions, is critical to understanding many chemical and biochemical processes. This is of special interest in the field of protein characterization through bottom-up proteomics, where peptide fragmentation spectra are used to identify proteins from complex mixtures. In peptide mass spectrometry, protonated peptides are generally fragmented by activating via collisions with a gaseous target to cause collision-induced dissociation (CID), which causes characteristic peptide fragmentation at the amide bond between amino acid residues. Fragment ions produced by a given activation method then provide sequence information through the ion types produced (e.g., N-terminal b and C-terminal y ions). The peptide fragmentation spectra are analyzed by peptide/protein identification algorithms. These algorithms compare experimental spectra to theoretical peptide spectra (or peak lists) generated from databases of protein sequences to identify the peptide sequence associated with the MS/MS spectrum. However, this approach is not without error; in fact, only a relatively small percentage of the peptide fragmentation spectra are correctly matched to peptide sequences. Many reasons exist to explain the discordance between the number of peptide MS/MS spectra generated and the number assigned to peptide sequences. First, only a small amount of chemical information is incorporated into the models used to generate the theoretical spectra. The simplified models of the peptide fragmentation process used by most peptide identification algorithms do not include nonstandard fragmentation pathways, including sequence scrambling as was reported by Paizs and co-workers, or the presence of isobaric nonstandard fragments, including the presence of non-C-terminal water loss, which was first reported by Gaskell and co-workers and more recently presented by Wysocki and co-workers for the model peptide system YAGFL. Additionally, currently available algorithms do not take full advantage of peptide fragment ion intensity information. Intensity enhancement can suggest preferential formation of certain fragment ions, providing additional certainty of the peptide sequence identification; without the incorporation of intensity, this information is Received: January 9, 2012 Revised: March 20, 2012 Published: March 26, 2012 Article