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A characteristic neutral loss of 44 Da is observed in the MS/MS spectra of Thr-containing sodiated peptides. A combination of tandem mass spectrometry and quantum chemical calculations calculated at the B3LYP/6-311G (d, p) level of ab initio theory is used to elucidate this fragmentation pathway. The high resolution mass spectrometry data indicate this neutral loss is acetaldehyde lost from the side chain of Thr rather than CO2. The intensity of this neutral loss can be enhanced when Thr residue is far from the C-terminus and when the C-terminus is esterified as well. The mechanism of the acetaldehyde loss is proposed to adopt a McLafferty-type rearrangement reaction, which involves a proton transfer from the hydroxyl of Thr side chain to its C-terminal neighboring carbonyl oxygen inducing the cleavage of the Ca–Cβ bond. This mechanism is further supported by examining the fragmentation of a [GT(tBu)G + Na]+ peptide derivative and by comparing the product ion spectra of [M + Na-44]+ of [GTGA + Na]+ with [M + Na]+ of [GGGA + Na]+. A similar neutral loss of HCHO can also be detected in Ser-containing peptides. Our computational results reveal that the most stable [GTG + Na]+ ion is present as a tridentate charge-solvated structure and the dissociation leading to the 44 loss is dynamically and energetically favorable. Copyright © 2015 John Wiley & Sons, Ltd.

A good understanding of gas-phase fragmentation chemistry of peptides is important for accurate protein identification. Additional product ions obtained by sodiated peptides can provide useful sequence information supplementary to protonated peptides and improve protein identification. In this work, we first demonstrate that the sodiated a3 ions are abundant in the tandem mass spectra of sodium-cationized peptides although observations of a3 ions have rarely been reported in protonated peptides. Quantum chemical calculations combined with tandem mass spectrometry are used to investigate this phenomenon by using a model tetrapeptide GGAG. Our results reveal that the most stable [a3 + Na − H]+ ion is present as a bidentate linear structure in which the sodium cation coordinates to the two backbone carbonyl oxygen atoms. Due to structural inflexibility, further fragmentation of the [a3 + Na − H]+ ion needs to overcome several relatively high energetic barriers to form [b2 + Na − H]+ ion with a diketopiperazine structure. As a result, low abundance of [b2 + Na − H]+ ion is detected at relatively high collision energy. In addition, our computational data also indicate that the common oxazolone pathway to generate [b2 + Na − H]+ from the [a3 + Na − H]+ ion is unlikely. The present work provides a mechanistic insight into how a sodium ion affects the fragmentation behaviors of peptides. Copyright © 2015 John Wiley & Sons, Ltd.

Peptide fragments such as b and y sequence ions generated upon low-energy collision-induced dissociation have been routinely used for tandem mass spectrometry (MS/MS)-based peptide/protein identification. The underlying formation mechanisms have been studied extensively and described within the literature. As a result, the 'mobile proton model' and 'pathways in competition model' have been built to interpret a majority of peptide fragmentation behavior. However, unusual peptide fragments which involve unfamiliar fragmentation pathways or various rearrangement reactions occasionally appear in MS/MS spectra, resulting in confused MS/MS interpretations. In this work, a series of unfamiliar c ions are detected in MS/MS spectra of the model peptides having an N-terminal Arg or deuterohemin group upon low-energy collision-induced dissociation process. Both the protonated Arg and deuterohemin group play an important role in retention of a positive charge at the N-terminus that is remote from the cleavage sites. According to previous reports and our studies involving amino acid substitutions and hydrogen-deuterium exchange, we propose a McLafferty-type rearrangement via charge-remote fragmentation as the potential mechanism to explain the formation of c ions from precursor peptide ions or unconventional b ions. Density functional theory calculations are also employed in order to elucidate the proposed fragmentation mechanisms. Copyright © 2016 John Wiley & Sons, Ltd.