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1. Top‐down mass spectrometry of intact phosphorylated β‐casein: Correlation between the precursor charge state and internal fragments

   https://doi.org/10.1002/jms.4364  

   Chen, Jianzhong ; Shiyanov, Pavel ; Green, Kari B. ( May 2019 , Journal of Mass Spectrometry)

   Phosphorylated proteins play essential roles in many cellular processes, and identification and characterization of the relevant phosphoproteins can help to understand underlying mechanisms. Herein, we report a collision‐induced dissociation top‐down approach for characterizing phosphoproteins on a quadrupole time‐of‐flight mass spectrometer. β‐casein, a protein with two major isoforms and five phosphorylatable serine residues, was used as a model. Peaks corresponding to intact β‐casein ions with charged states up to 36⁺were detected. Tandem mass spectrometry was performed on β‐casein ions of different charge states (12⁺, and 15⁺to 28⁺) in order to determine the effects of charge state on dissociation of this protein. Most of the abundant fragments corresponded to y, b ions, and internal fragments caused by cleavage of the N‐terminal amide bond adjacent to proline residues (Xxx‐Pro). The abundance of internal fragments increased with the charge state of the protein precursor ion; these internal fragments predominantly arose from one or two Xxx‐Pro cleavage events and were difficult to accurately assign. The presence of abundant sodium adducts of β‐casein further complicated the spectra. Our results suggest that when interpreting top‐down mass spectra of phosphoproteins and other proteins, researchers should consider the potential formation of internal fragments and sodium adducts for reliable characterization.

    

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2. A plethora of isomerization processes and hydrogen scrambling in the fragmentation of the methanol dimer cation: a PEPICO study

   https://doi.org/10.1039/d1cp05155e  

   Wu, Xiangkun ; Zhou, Xiaoguo ; Bjelić, Saša ; Hemberger, Patrick ; Sztáray, Bálint ; Bodi, Andras ( January 2022 , Physical Chemistry Chemical Physics)

   The valence photoionization of light and deuterated methanol dimers was studied by imaging photoelectron photoion coincidence spectroscopy in the 10.00–10.35 eV photon energy range. Methanol clusters were generated in a rich methanol beam in nitrogen after expansion into vacuum. They generally photoionize dissociatively to protonated methanol cluster cations, (CH 3 OH) n H + . However, the stable dimer parent ion (CH 3 OH) 2 + is readily detected below the dissociation threshold to yield the dominant CH 3 OH 2 + fragment ion. In addition to protonated methanol, we could also detect the water- and methyl-loss fragment ions of the methanol dimer cation for the first time. These newly revealed fragmentation channels are slow and cannot compete with protonated methanol cation formation at higher internal energies. In fact, the water- and methyl-loss fragment ions appear together and disappear at a ca. 150 meV higher energy in the breakdown diagram. Experiments with selectively deuterated methanol samples showed H scrambling involving two hydroxyl and one methyl hydrogens prior to protonated methanol formation. These insights guided the potential energy surface exploration to rationalize the dissociative photoionization mechanism. The potential energy surface was further validated by a statistical model including isotope effects to fit the experiment for the light and the perdeuterated methanol dimers simultaneously. The (CH 3 OH) 2 + parent ion dissociates via five parallel channels at low internal energies. The loss of both CH 2 OH and CH 3 O neutral fragments leads to protonated methanol. However, the latter, direct dissociation channel is energetically forbidden at low energies. Instead, an isomerization transition state is followed by proton transfer from a methyl group, which leads to the CH 3 (H)OH + ⋯CH 2 OH ion, the precursor to the CH 2 OH-, H 2 O-, and CH 3 -loss fragments after further isomerization steps, in part by a roaming mechanism. Water loss yields the ethanol cation, and two paths are proposed to account for m/z 49 fragment ions after CH 3 loss. The roaming pathways are quickly outcompeted by hydrogen bond breaking to yield CH 3 OH 2 + , which explains the dominance of the protonated methanol fragment ion in the mass spectrum. 

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3. Charge transfer dissociation of a branched glycan with alkali and alkaline earth metal adducts

   https://doi.org/10.1002/jms.4774  

   Sasiene, Zachary J. ; Ropartz, David ; Rogniaux, Hélène ; Jackson, Glen P. ( June 2021 , Journal of Mass Spectrometry)

   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.

    

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4. Differentiating aspartic acid isomers and epimers with charge transfer dissociation mass spectrometry (CTD-MS)

   https://doi.org/10.1039/D1AN02279B  

   Edwards, Halle M. ; Wu, Hoi-Ting ; Julian, Ryan R. ; Jackson, Glen P. ( March 2022 , The Analyst)

   The ability to understand the function of a protein often relies on knowledge about its detailed structure. Sometimes, seemingly insignificant changes in the primary structure of a protein, like an amino acid substitution, can completely disrupt a protein's function. Long-lived proteins (LLPs), which can be found in critical areas of the human body, like the brain and eye, are especially susceptible to primary sequence alterations in the form of isomerization and epimerization. Because long-lived proteins do not have the corrective regeneration capabilities of most other proteins, points of isomerism and epimerization that accumulate within the proteins can severely hamper their functions and can lead to serious diseases like Alzheimer's disease, cancer and cataracts. Whereas tandem mass spectrometry (MS/MS) in the form of collision-induced dissociation (CID) generally excels at peptide characterization, MS/MS often struggles to pinpoint modifications within LLPs, especially when the differences are only isomeric or epimeric in nature. One of the most prevalent and difficult-to-identify modifications is that of aspartic acid between its four isomeric forms: l -Asp, l -isoAsp, d -Asp, and d -isoAsp. In this study, peptides containing isomers of Asp were analyzed by charge transfer dissociation (CTD) mass spectrometry to identify spectral features that could discriminate between the different isomers. For the four isomers of Asp in three model peptides, CTD produced diagnostic ions of the form c n +57 on the N-terminal side of iso-Asp residues, but not on the N-terminal side of Asp residues. Using CTD, the l - and d forms of Asp and isoAsp could also be differentiated based on the relative abundance of y - and z ions on the C-terminal side of Asp residues. Differentiation was accomplished through a chiral discrimination factor, R , which compares an ion ratio in a spectrum of one epimer or isomer to the same ion ratio in the spectrum of a different epimer or isomer. The R values obtained using CTD are as robust and statistically significant as other fragmentation techniques, like radical directed dissociation (RDD). In summary, the extent of backbone and side-chain fragments produced by CTD enabled the differentiation of isomers and epimers of Asp in a variety of peptides. 

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5. Intrinsic reactivity of [OUCH] + : Apparent synthesis of [OUS] + by reaction with CS 2

   https://doi.org/10.1002/rcm.9260  

   Metzler, Luke J. ; Farmen, Christopher T. ; Fry, Allison N. ; Seibert, Mark P. ; Massari, Kayla A. ; Corcovilos, Theodore A. ; van Stipdonk, Michael J. ( February 2022 , Rapid Communications in Mass Spectrometry)

   Building on our report that collision‐induced dissociation (CID) can be used to create the highly reactive U‐alkylidyne species [O=U≡CH]⁺, our goal was to determine whether the species could be as an intermediate for synthesis of [OUS]⁺by reaction with carbon disulfide (CS₂).

   Cationic uranyl‐propiolate precursor ions were generated by electrospray ionization, and multiple‐stage CID in a linear trap instrument was used to prepare [O=U≡CH]⁺. Neutral CS₂was admitted into the trap through a modified He inlet and precision leak valves.

   The [O=U≡CH]⁺ion reacts with CS₂to generate [OUS]⁺. CID of [OUS]⁺causes elimination of the axial sulfide ligand to generate [OU]⁺. Using isotopically labeled reagent, we found that [OUS]⁺reacts with O₂to create [UO₂]⁺.

   [O=U≡CH]⁺proves to be a useful reagent ion for synthesis of [OUS]⁺, a species that to date has only been created by gas‐phase reactions of U⁺and U²⁺. Dissociation of [OUS]⁺to create [OU]⁺, but not [US]⁺, and the efficient conversion of the species into [UO₂]⁺, is consistent with the relative differences in U–O and U–S bond energies.

    

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