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1. Salt-Bridged Peptide Anion Gaseous Structures Enable Efficient Negative Ion Electron Capture Dissociation

   https://doi.org/10.1021/jasms.4c00030  

   Wang, Ning; Dixit, Sugyan M; Lee, Teresa; DeFiglia, Steven A; Ruotolo, Brandon T; Håkansson, Kristina (April 2024, Journal of the American Society for Mass Spectrometry)

   We previously discovered that electron attachment to gaseous peptide anions can occur within a relatively narrow electron energy range. The resulting charge-increased radical ions undergo dissociation analogous to conventional cation electron capture/transfer dissociation (ECD/ETD), thus enabling a novel tandem mass spectrometry (MS/MS) technique that we termed negative ion electron capture dissociation (niECD). We proposed that gaseous zwitterionic structures are required for niECD with electron capture either occurring at or being directed by a positively charged site. Here, we further evaluate this zwitterion mechanism by performing niECD of peptides derivatized to alter their ability to form zwitterionic gaseous structures. Introduction of a fixed positive charge tag, a highly basic guanidino group, or a highly acidic sulfonate group to promote zwitterionic structures in singly charged anions, rescued the niECD ability of a peptide refractory to niECD in its unmodified form. We also performed a systematic study of five sets of synthetic peptides with decreasing zwitterion propensity and found that niECD efficiency decreased accordingly, further supporting the zwitterion mechanism. However, traveling-wave ion mobility-mass spectrometry experiments, performed to gain further insight into the gas-phase structures of peptides showing high niECD efficiency, exhibited an inverse correlation between the orientationally averaged collision cross sections and niECD efficiency. These results indicate that compact salt-bridged structures are also a requirement for effective niECD. 

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2. Are Internal Fragments Observable in Electron Based Top-Down Mass Spectrometry?

   https://doi.org/10.1016/j.mcpro.2024.100814  

   Mikawy, Neven N; Rojas_Ramírez, Carolina; DeFiglia, Steven A; Szot, Carson W; Le, Jessie; Lantz, Carter; Wei, Benqian; Zenaidee, Muhammad A; Blakney, Greg T; Nesvizhskii, Alexey I; et al (September 2024, Molecular & Cellular Proteomics)

   Protein tandem mass spectrometry (MS/MS) often generates sequence-informative fragments from backbone bond cleavages near the termini. This lack of fragmentation in the protein interior is particularly apparent in native top-down MS. Improved sequence coverage, critical for reliable annotation of posttranslational modifications (PTMs) and sequence variants, may be obtained from internal fragments generated by multiple backbone cleavage events. However, internal fragment assignments can be error prone due to isomeric/isobaric fragments from different parts of a protein sequence. Also, internal fragment generation propensity depends on the chosen MS/MS activation strategy. Here, we examine internal fragment formation in electron capture dissociation (ECD) and electron transfer dissociation (ETD) following native and denaturing MS, as well as liquid chromatography (LC)/MS of several proteins. Experiments were undertaken on multiple instruments, including Q-ToF, Orbitrap, and high-field FT-ICR across four laboratories. ECD was performed at both ultrahigh vacuum and at similar pressure to ETD conditions. Two complementary software packages were used for data analysis. When feasible, ETD-higher-energy collision dissociation (ETD-HCD) MS3 was performed to validate/refute potential internal fragment assignments, including differentiating MS3 fragmentation behavior of radical vs. even-electron primary fragments. We show that, under typical operating conditions, internal fragments cannot be confidently assigned in ECD, nor ETD. On the other hand, such fragments, along with some b-type terminal fragments (not typically observed in ECD/ETD spectra) appear at atypical ECD operating conditions, suggesting they originate from a separate ion-electron activation process. Furthermore, atypical fragment ion types, e.g., x ions, are observed at such conditions as well as upon EThcD, presumably due to vibrational activation of radical z-type ions. 

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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)

   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. 

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4. Resolving Isomers of Star-Branched Poly(Ethylene Glycols) by IMS-MS Using Multiply Charged Ions

   https://doi.org/10.1021/jasms.0c00045  

   Austin, Calvin A.; Inutan, Ellen D.; Bohrer, Brian C.; Li, Jing; Fischer, Joshua L.; Wijerathne, Kanchana; Foley, Casey D.; Lietz, Christopher B.; Woodall, Daniel W.; Imperial, Lorelie F.; et al (January 2020, Journal of the American Society for Mass Spectrometry)

   null (Ed.)

   Ion mobility spectrometry (IMS) mass spectrometry (MS) centers on the ability to separate gaseous structures by size, charge, shape, and followed by mass-to-charge (m/z). For oligomeric structures, improved separation is hypothesized to be related to the ability to extend structures through repulsive forces between cations electrostatically bonded to the oligomers. Here we show the ability to separate differently branched multiply charged ions of star-branched poly(ethylene glycol) oligomers (up to 2000 Da) regardless of whether formed by electrospray ionization (ESI) charged solution droplets or from charged solid particles produced directly from a surface by matrix-assisted ionization. Detailed structural characterization of isomers of the star-branched compositions was first established using a home-built high-resolution ESI IMS-MS instrument. The doubly charged ions have well-resolved drift times, achieving separation of isomers and also allowing differentiation of star-branched versus linear oligomers. An IMS-MS “snapshot” approach allows visualization of architectural dispersity and (im)purity of samples in a straightforward manner. Analyses capabilities are shown for different cations and ionization methods using commercially available traveling wave IMS-MS instruments. Analyses directly from surfaces using the new ionization processes are, because of the multiply charging, not only associated with the benefits of improved gas-phase separations, relative to that of ions produced by matrix-assisted laser desorption/ionization, but also provide the potential for spatially resolved measurements relative to ESI and other ionization methods. 

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5. Solution- and gas-phase behavior of decavanadate: implications for mass spectrometric analysis of redox-active polyoxidometalates

   https://doi.org/10.1039/d1qi01618k  

   Favre, Daniel; Bobst, Cedric E.; Eyles, Stephen J.; Murakami, Heide; Crans, Debbie C.; Kaltashov, Igor A. (March 2022, Inorganic Chemistry Frontiers)

   Decavanadate (V 10 O 28 6− or V10) is a paradigmatic member of the polyoxidometalate (POM) family, which has been attracting much attention within both materials/inorganic and biomedical communities due to its unique structural and electrochemical properties. In this work we explored the utility of high-resolution electrospray ionization (ESI) mass spectrometry (MS) and ion exclusion chromatography LC/MS for structural analysis of V10 species in aqueous solutions. While ESI generates abundant molecular ions representing the intact V10 species, their isotopic distributions show significant deviations from the theoretical ones. A combination of high-resolution MS measurements and hydrogen/deuterium exchange allows these deviations to be investigated and interpreted as a result of partial reduction of V10. While the redox processes are known to occur in the ESI interface and influence the oxidation state of redox-active analytes, the LC/MS measurements using ion exclusion chromatography provide unequivocal evidence that the mixed-valence V10 species exist in solution, as extracted ion chromatograms representing V10 molecular ions at different oxidation states exhibit distinct elution profiles. The spontaneous reduction of V10 in solution is seen even in the presence of hydrogen peroxide and has not been previously observed. The susceptibility to reduction of V10 is likely to be shared by other redox active POMs. In addition to the molecular V10 ions, a high-abundance ionic signal for a V 10 O 26 2− anion was displayed in the negative-ion ESI mass spectra. None of the V 10 O 26 cations were detected in ESI MS, and only a low-abundance signal was observed for V 10 O 26 anions with a single negative charge, indicating that the presence of abundant V 10 O 26 2− anions in ESI MS reflects gas-phase instability of V 10 O 28 anions carrying two charges. The gas-phase origin of the V 10 O 26 2− anion was confirmed in tandem MS measurements, where mild collisional activation was applied to V10 molecular ions with an even number of hydrogen atoms (H 4 V 10 O 28 2− ), resulting in a facile loss of H 2 O molecules and giving rise to V 10 O 26 2− as the lowest-mass fragment ion. Water loss was also observed for V 10 O 28 anions carrying an odd number of hydrogen atoms ( e.g. , H 5 V 10 O 28 − ), followed by a less efficient and incomplete removal of an OH˙ radical, giving rise to both HV 10 O 26 − and V 10 O 25 − fragment ions. Importantly, at least one hydrogen atom was required for ion fragmentation in the gas phase, as no further dissociation was observed for any hydrogen-free V10 ionic species. The presented workflow allows a distinction to be readily made between the spectral features revealing the presence of non-canonical POM species in the bulk solution from those that arise due to physical and chemical processes occurring in the ESI interface and/or the gas phase. 

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