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1. Understanding the structural complexity of dissolved organic matter: isomeric diversity

   https://doi.org/10.1039/c8fd00221e  

   Leyva, Dennys ; Tose, Lilian V. ; Porter, Jacob ; Wolff, Jeremy ; Jaffé, Rudolf ; Fernandez-Lima, Francisco ( August 2019 , Faraday Discussions)

   In the present work, the advantages of ESI-TIMS-FT-ICR MS to address the isomeric content of dissolved organic matter are studied. While the MS spectra allowed the observation of a high number of peaks ( e.g. , PAN-L: 5004 and PAN-S: 4660), over 4× features were observed in the IMS-MS domain ( e.g. , PAN-L: 22 015 and PAN-S: 20 954). Assuming a total general formula of C x H y N 0–3 O 0–19 S 0–1 , 3066 and 2830 chemical assignments were made in a single infusion experiment for PAN-L and PAN-S, respectively. Most of the identified chemical compounds (∼80%) corresponded to highly conjugated oxygen compounds (O 1 –O 20 ). ESI-TIMS-FT-ICR MS provided a lower estimate of the number of structural and conformational isomers ( e.g. , an average of 6–10 isomers per chemical formula were observed). Moreover, ESI-q-FT-ICR MS/MS at the level of nominal mass ( i.e. , 1 Da isolation) allowed for further estimation of the number of isomers based on unique fragmentation patterns and core fragments; the later suggested that multiple structural isomers could have very closely related CCS. These studies demonstrate the need for ultrahigh resolution TIMS mobility scan functions ( e.g. , R = 200–500) in addition to tandem MS/MS isolation strategies. 

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2. Metal ions induced secondary structure rearrangements: mechanically interlocked lasso vs. unthreaded branched-cyclic topoisomers

   https://doi.org/10.1039/c8an00138c  

   Jeanne Dit Fouque, Kevin ; Moreno, Javier ; Hegemann, Julian D. ; Zirah, Séverine ; Rebuffat, Sylvie ; Fernandez-Lima, Francisco ( January 2018 , The Analyst)

   Metal ions can play a significant role in a variety of important functions in protein systems including cofactor for catalysis, protein folding, assembly, structural stability and conformational change. In the present work, we examined the influence of alkali (Na, K and Cs), alkaline earth (Mg and Ca) and transition (Co, Ni and Zn) metal ions on the conformational space and analytical separation of mechanically interlocked lasso peptides. Syanodin I, sphingonodin I, caulonodin III and microcin J25, selected as models of lasso peptides, and their respective branched-cyclic topoisomers were submitted to native nESI trapped ion mobility spectrometry-mass spectrometry (TIMS-MS). The high mobility resolving power of TIMS permitted to group conformational families regardless of the metal ion. The lower diversity of conformational families for syanodin I as compared to the other lasso peptides supports that syanodin I probably forms tighter binding interactions with metal ions limiting their conformational space in the gas-phase. Conversely, the higher diversity of conformational families for the branched-cyclic topologies further supports that the metal ions probably interact with a higher number of electronegative groups arising from the fully unconstraint C-terminal part. A correlation between the lengths of the loop and the C-terminal tail with the conformational space of lasso peptides becomes apparent upon addition of metal ions. It was shown that the threaded C-terminal region in lasso peptides allows only for distinct interactions of the metal ion with either residues in the loop or tail region. This limits the size of the interacting region and apparently leads to a bias of metal ion binding in either the loop or tail region, depending whichever section is larger in the respective lasso peptide. For branched-cyclic peptides, the non-restricted C-terminal tail allows metal coordination by residues throughout this region, which can result in gas-phase structures that are sometimes even more compact than the lasso peptides. The high TIMS resolution also resulted in the separation of almost all lasso and branched-cyclic topoisomer metal ions ( r ∼ 2.1 on average). It is also shown that the metal incorporation ( e.g. , doubly cesiated species) can lead to the formation of a simplified IMS pattern (or preferential conformers), which results in baseline analytical separation and discrimination between lasso and branched-cyclic topologies using TIMS-MS. 

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3. Capillary Zone Electrophoresis-Electron-Capture Collision-Induced Dissociation on a Quadrupole Time-of-Flight Mass Spectrometer for Top-Down Characterization of Intact Proteins

   https://doi.org/10.1021/jasms.0c00484  

   Shen, Xiaojing ; Xu, Tian ; Hakkila, Blake ; Hare, Mike ; Wang, Qianjie ; Wang, Qianyi ; Beckman, Joseph S. ; Sun, Liangliang ( March 2021 , Journal of the American Society for Mass Spectrometry)

   null (Ed.)

   Mass spectrometry (MS)-based denaturing top-down proteomics (dTDP) requires high-capacity separation and extensive gas-phase fragmentation of proteoforms. Herein, we coupled capillary zone electrophoresis (CZE) to electron-capture collision-induced dissociation (ECciD) on an Agilent 6545 XT quadrupole time-of-flight (Q-TOF) mass spectrometer for dTDP for the first time. During ECciD, the protein ions were first fragmented using ECD, followed by further activation and fragmentation by applying a CID potential. In this pilot study, we optimized the CZE-ECciD method for small proteins (lower than 20 kDa) regarding the charge state of protein parent ions for fragmentation and the CID potential applied to maximize the protein backbone cleavage coverage and the number of sequence-informative fragment ions. The CZE-ECciD Q-TOF platform provided extensive backbone cleavage coverage for three standard proteins lower than 20 kDa from only single charge states in a single CZE-MS/MS run in the targeted MS/MS mode, including ubiquitin (97%, +7, 8.6 kDa), superoxide dismutase (SOD, 87%, +17, 16 kDa), and myoglobin (90%, +16, 17 kDa). The CZE-ECciD method produced comparable cleavage coverage of small proteins (i.e., myoglobin) with direct-infusion MS studies using electron transfer dissociation (ETD), activated ion-ETD, and combinations of ETD and collision-based fragmentation on high-end orbitrap mass spectrometers. The results render CZE-ECciD a new tool for dTDP to enhance both separation and gas-phase fragmentation of proteoforms. 

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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. Tandem trapped ion mobility spectrometry

   https://doi.org/10.1039/c7an02054f  

   Liu, Fanny C. ; Ridgeway, Mark E. ; Park, Melvin A. ; Bleiholder, Christian ( January 2018 , The Analyst)

   There is currently a strong interest in the use of ion mobility spectrometry-mass spectrometry (IMS-MS) instrumentation for structural biology. In these applications, momentum transfer cross sections derived from IMS-MS measurements are used to reconstruct the three-dimensional analyte structure. Recent reports indicate that additional structural information can be extracted from measuring changes in cross sections in response to changes of the analyte structure. To further this approach, we constructed a tandem trapped IMS analyser (TIMS-TIMS) and incorporated it in a QqTOF mass spectrometer. TIMS-TIMS is constructed by coupling two TIMS analysers via an “interface region” composed of two apertures. We show that peptide oligomers (bradykinin) and native-like protein (ubiquitin) ions can be preserved through the course of an experiment in a TIMS-TIMS analyser. We demonstrate the ability to collisionally-activate as well as to trap mobility-selected ions, followed by subsequent mobility-analysis. In addition to inducing conformational changes, we show that we can fragment low charge states of ubiquitin at >1 mbar between the TIMS analysers with significant sequence coverage. Many fragment ions exhibit multiple features in their TIMS spectra, which means that they may not generally exist as the most stable isomer. The ability of TIMS-TIMS to dissociate mobility-selected protein ions and to measure the cross sections of their fragment ions opens new possibilities for IMS-based structure elucidation. 

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