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RationaleThe function of a protein or the binding affinity of an antibody can be substantially altered by the replacement of leucine (Leu) with isoleucine (Ile), and vice versa, so the ability to identify the correct isomer using mass spectrometry can help resolve important biological questions. Tandem mass spectrometry approaches for Leu/Ile (Xle) discrimination have been developed, but they all have certain limitations. MethodsFour model peptides and two wild‐type peptide sequences containing either Leu or Ile residues were subjected to charge transfer dissociation (CTD) mass spectrometry on a modified three‐dimensional ion trap. The peptides were analyzed in both the 1+ and 2+ charge states, and the results were compared to conventional collision‐induced dissociation spectra of the same peptides obtained using the same instrument. ResultsCTD resulted in 100% sequence coverage for each of the studied peptides and provided a variety of side‐chain cleavages, includingd,wandvions. Using CTD, reliabledandwions of Xle residues were observed more than 80% of the time. When present,dions are typically greater than 10% of the abundance of the correspondingaions from which they derive, andwions are typically more abundant than thezions from which they derive. ConclusionsCTD has the benefit of being applicable to both 1+ and 2+ precursor ions, and the overall performance is comparable to that of other high‐energy activation techniques like hot electron capture dissociation and UV photodissociation. CTD does not require chemical modifications of the precursor peptides, nor does it require additional levels of isolation and fragmentation. 

Abstract Alkali and alkaline earth metal adducts of a branched glycan, XXXG, were analyzed with helium charge transfer dissociation (He‐CTD) and low‐energy collision‐induced dissociation (LE‐CID) to investigate if metalation would impact the type of fragments generated and the structural characterization of the analyte. The studied adducts included 1+ and 2+ precursors involving one or more of the cations: H⁺, Na⁺, K⁺, Ca²⁺, and Mg²⁺. Regardless of the metal adduct, He‐CTD generated abundant and numerous glycosidic and cross‐ring cleavages that were structurally informative and able to identify the 1,4‐linkage and 1,6‐branching patterns. In contrast, the LE‐CID spectra mainly contained glycosidic cleavages, consecutive fragments, and numerous neutral losses, which complicated spectral interpretation. LE‐CID of [M + K + H]²⁺and [M + Na]⁺precursors generated a few cross‐ring cleavages, but they were not sufficient to identify the 1,4‐linkage and 1,6‐branching pattern of the XXXG xyloglucan. He‐CTD predominantly generated 1+ fragments from 1+ precursors and 2+ product ions from 2+ precursors, although both LE‐CID and He‐CTD were able to generate 1+ product ions from 2+ adducts of magnesium and calcium. The singly charged fragments derive from the loss of H⁺from the metalated product ions and the formation of a protonated complementary product ion; such observations are similar to previous reports for magnesium and calcium salts undergoing electron capture dissociation (ECD) activation. However, during He‐CTD, the [M + Mg]²⁺precursor generated more singly charged product ions than [M + Ca]²⁺, either because Mg has a higher second ionization potential than Ca or because of conformational differences and the locations of the charging adducts during fragmentation. He‐CTD of the [M + 2Na]²⁺and the [M + 2 K]²⁺precursors generated singly charged product ions from the loss of a sodium ion and potassium ion, respectively. In summary, although the metal ions influence the mass and charge state of the observed product ions, the metal ions had a negligible effect on the types of cross‐ring cleavages observed.