Experimental Measurements of Relative Mobility Shifts Resulting from Isotopic Substitutions with High-Resolution Cyclic Ion Mobility Separations

Abstract

Herein, we report on the experimental measurements for estimated relative mobility shifts caused by changes in mass distribution from isotopic substitutions in isotopologues and isotopomers with high-resolution cyclic ion mobility separations. By utilizing unlabeled and fully labeled isotopologues with the same isotopic substitutions (i.e., 2H or 13C), we created a highly precise mobility scale for each set analyzed to determine the magnitude of such mass distribution shifts and thus calculate estimated deviations from expected, theoretical reduced mass contributions. We observed relative mobility shifts in various isotopologues (e.g., hexadecyltrimethylammonium, sucrose, and palmitic acid species) that deviated from reduced mass theory, according to the Mason–Schamp relationship, ranging in estimated magnitude from ∼0.007% up to ∼0.1% in relative mobility. More interestingly, it was found that two deuterated palmitic acid isotopomers also differed by ∼0.03% from one another in their respective relative mobility shifts. Our results are the first report of isotopologue and isotopomer separations on a commercially available cyclic ion mobility spectrometry-mass spectrometry platform. We envision that our presented mobility scale methodology will have broad applicability in studying the effect of mass distribution changes from isotopic substitutions in other biomolecules and help pave the way for the improvement of ion mobility theory and collision cross section calculators.

Isomer and Conformer-Specific Mass Distribution-Based Isotopic Shifts in High-Resolution Cyclic Ion Mobility Separations

“…values (Equation ), were highly precise and pathlength-independent (Figure ). Additionally, based on our previous work, we do not believe that varying TW conditions will lead to different results than those presented in this work, although further study is needed to fully assess this . We were able to demonstrate mass distribution-based shifts not only for Br and Cl-containing compounds but also for C-containing ones, which are more practical for biologically relevant compounds.…”

“…Additionally, based on our previous work, we do not believe that varying TW conditions will lead to different results than those presented in this work, although further study is needed to fully assess this. 44 We were able to demonstrate mass distribution-based shifts not only for Br and Cl-containing compounds but also for C-containing ones, which are more practical for biologically relevant compounds. It is relevant to note that in all experiments presented in this work, the arrival time order matched that of the order of magnitude of the mass distribution-based shifts (i.e., the lower mobility species always produced greater t Rel.…”

“…Recently, our group has experimentally estimated the magnitude of such mass distribution-based shifts on a commercially available cyclic ion mobility spectrometry-mass spectrometry (cIMS-MS) platform. 44 By using isotopically labeled calibrants to create a relative mobility scale for hexadecyltrimethylammonium, sucrose, and palmitic acid isotopologues and isotopomers, we could assess the deviations from theoretical reduced mass contributions and thus calculate the estimated relative mobility shifts from mass distributionbased effects. 44 Our results raised the question if these mass distribution-based shifts could be observed in more realistic, and biologically relevant, samples such as isomers and conformers.…”

“…44 By using isotopically labeled calibrants to create a relative mobility scale for hexadecyltrimethylammonium, sucrose, and palmitic acid isotopologues and isotopomers, we could assess the deviations from theoretical reduced mass contributions and thus calculate the estimated relative mobility shifts from mass distributionbased effects. 44 Our results raised the question if these mass distribution-based shifts could be observed in more realistic, and biologically relevant, samples such as isomers and conformers. Specifically, if such shifts could be observed among the naturally occurring isotopologues present in a given molecule's isotopic envelope (e.g., 13 C, 81 Br, 37 Cl, etc), it could potentially open new avenues into exploring mass distribution as an added dimension to an IMS-MS-based experiment.…”

Evaluating the Utility of Temporal Compression in High-Resolution Traveling Wave-Based Cyclic Ion Mobility Separations

“…Since IMS resolution scales approximately by the square root of the separation pathlength (e.g., a 100 m separation would provide 10 times higher resolution than a 1 m separation), longer IMS separations are very attractive for resolving very structurally similar molecules, such as cis/trans lipid isomers, peptide epimers, oligosaccharide anomers, and isotopologues/isotopomers. ,− These longer pathlength implementations are made possible by the lower voltage requirements in TWIMS and not possible in other IMS modalities, such as the large voltage drop required in DTIMS to achieve even a few meters of separation. − Currently, two commercially available traveling wave-based IMS-MS platforms (structures for lossless ion manipulations, MOBIE, from MOBILion, and the Select Series Cyclic IMS, cIMS, from Waters Corporation) have exploited this and achieved separation pathlengths >10 m and up to 2 km in a homebuilt platform. , Both SLIM IMS-MS and cIMS-MS utilize traveling wave separations, giving added flexibility and customization for experiments (e.g., IMS/IMS, peak selection, etc. ).…”