Dataset Information

Quantitative Analysis of Diubiquitin Isomers Using Ion Mobility Mass Spectrometry.

ABSTRACT: The diversity of ubiquitin modifications calls for methods to better characterize ubiquitin chain linkage, length, and morphology. Here, we use multiple linear regression analysis coupled with ion mobility mass spectrometry (IM-MS) to quantify the relative abundance of different ubiquitin dimer isomers. We demonstrate the utility and robustness of this approach by quantifying the relative abundance of different ubiquitin dimers in complex mixtures and comparing the results to the standard, bottom-up ubiquitin AQUA method. Our results provide a foundation for using multiple linear regression analysis and IM-MS to characterize more complex ubiquitin chain architectures.

SUBMITTER: Shestoperova EI

PROVIDER: S-EPMC10712319 | biostudies-literature | 2023 May

REPOSITORIES: biostudies-literature

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Quantitative Analysis of Diubiquitin Isomers Using Ion Mobility Mass Spectrometry.

Shestoperova Elizaveta I EI Ivanov Daniil G DG Strieter Eric R ER

Journal of the American Society for Mass Spectrometry 20230404 5

The diversity of ubiquitin modifications calls for methods to better characterize ubiquitin chain linkage, length, and morphology. Here, we use multiple linear regression analysis coupled with ion mobility mass spectrometry (IM-MS) to quantify the relative abundance of different ubiquitin dimer isomers. We demonstrate the utility and robustness of this approach by quantifying the relative abundance of different ubiquitin dimers in complex mixtures and comparing the results to the standard, botto ...[more]

PMID: 37014729

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Project description:RationaleCoordinatively driven self-assembly of transition metal ions and bidentate ligands gives rise to organometallic complexes that usually contain superimposed isobars, isomers, and conformers. In this study, the double dispersion ability of ion mobility mass spectrometry (IM-MS) was used to provide a comprehensive structural characterization of the self-assembled supramolecular complexes by their mass and charge, revealed by the MS event, and their shape and collision cross-section (Ω), revealed by the IM event.MethodsSelf-assembled complexes were synthesized by reacting a bis(terpyridine) ligand exhibiting a 60o dihedral angle between the two ligating terpyridine sites (T) with divalent Zn, Ni, Cd, or Fe. The products were isolated as (Metal2+ [T])n (PF6 )2n salts and analyzed using IM-MS after electrospray ionization (ESI) which produced several charge states from each n-mer, depending on the number of PF6 - anions lost upon ESI. Experimental Ω data, derived using IM-MS, and computational Ω predictions were used to elucidate the size and architecture of the complexes.ResultsOnly macrocyclic dimers, trimers, and tetramers were observed with Cd2+ , whereas Zn2+ formed the same plus hexameric complexes. These two metals led to the simplest product distributions and no linear isomers. In sharp contrast, Ni2+ and Fe2+ formed all possible ring sizes from dimer to hexamer as well as various linear isomers. The experimental and theoretical Ω data indicated rather planar macrocyclic geometries for the dimers and trimers, twisted 3D architectures for the larger rings, and substantially larger sizes with spiral conformation for the linear congeners. Adding PF6 - to the same complex was found to mainly cause size contraction due to new stabilizing anion-cation interactions.ConclusionsComplete structural identification could be accomplished using ESI-IM-MS. Our results affirm that self-assembly with Cd2+ and Zn2+ proceeds through reversible equilibria that generate the thermodynamically most stable structures, encompassing exclusively macrocyclic architectures that readily accommodate the 60o ligand used. In contrast, complexation with Ni2+ and Fe2+ , which form stronger coordinative bonds, proceeds through kinetic control, leading to more complex mixtures and kinetically trapped less stable architectures, such as macrocyclic pentamers and linear isomers.

Project description:IntroductionIon mobility-mass spectrometry (IM-MS) is an emerging technique in the -omics fields that has broad potential applicability to the clinical lab. As a rapid, gas-phase structure-based separation technique, IM-MS offers promise in isomer separations and can be easily combined with existing LC-MS methods (i.e., LC-IM-MS). Several experimental conditions, including analyte cation adducts and drift composition further provide a means to tune separations for global and/or targeted applications.ObjectivesThe primary objective of this study was to demonstrate the utility of IM-MS under a range of experimental conditions for detection of glucocorticoids, and specifically for the separation of several isomeric pairs.MethodsLC-IM-MS was used to characterize 16 glucocorticoids including three isomer pairs: cortisone/prednisolone, betamethasone/dexamethasone, and flunisolide/triamcinolone acetonide. Collision cross section (CCS) values were measured for all common adducts (e.g., protonated and sodiated) using both step-field and single-field methods. Alternative alkali, alkaline earth, and transition metals were introduced, such that their adducts could also be measured. Finally, four different drift gases (helium, nitrogen, argon, and carbon dioxide) were compared for their relative separation capability.ResultsLC-IM-MS offered a robust, multidimensional separation technique that allowed for the 16 glucocorticoids to be analyzed and separated in three-dimensions (retention time, CCS, and m/z). Despite the relatively modest resolution of isomer pairs under standard conditions (i.e., nitrogen drift gas, sodiated ions, etc.), improvements were observed for alkaline earth and transition metals (notable barium adducts) and in carbon dioxide drift gas.ConclusionIn summary, LC-IM-MS offers potential as a clinical method due to its ease of coupling with traditional LC-MS methods and its promise for tuning separations to better resolve targeted and/or global isomers in complex biological samples.

Dataset Information

Quantitative Analysis of Diubiquitin Isomers Using Ion Mobility Mass Spectrometry.

Publications

Quantitative Analysis of Diubiquitin Isomers Using Ion Mobility Mass Spectrometry.

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