Ultrahigh-Resolution Ion-Mobility Spectrometry Research Articles

Analyzing glycans cleaved from a biotherapeutic protein using ultrahigh-resolution ion mobility spectrometry together with cryogenic ion spectroscopy.

Glycans covalently attached to protein biotherapeutics have a significant impact on their biological activity, clearance, and safety. As a result, glycosylation is categorized as a critical quality attribute that needs an adequate analytical approach to guarantee product quality. However, the isomeric complexity and branched structure of glycans makes their analysis a significant challenge. In this work, we propose a multidimensional approach for monitoring released glycans that combines ultrahigh-resolution ion mobility spectrometry (IMS) and cryogenic vibrational spectroscopy, and we demonstrate this technique by characterizing four N-glycans cleaved from the therapeutic fusion protein etanercept that range in abundance from 1% to 22% of the total N-glycan content. The recorded vibrational spectra exhibit well-resolved transitions that can be used as a fingerprint to identify a particular glycan. This work represents an important advance in the analysis of N-linked glycans cleaved from biopharmaceutical proteins that could eventually be used as tool for monitoring biopharmaceutical glycoforms.

Separation and Identification of Glycan Anomers Using Ultrahigh-Resolution Ion-Mobility Spectrometry and Cryogenic Ion Spectroscopy.

The analysis of carbohydrates, or glycans, is challenging for established structure-sensitive gas-phase methods. The multitude of possible stereo-, regio-, and structural isomers makes them substantially more complex to analyze than DNA or proteins, and no one method is currently able to fully resolve them. While the combination of tandem mass spectrometry (MS) and ion-mobility spectrometry (IMS) have made important inroads in glycan analysis, in many cases, this approach is still not able to identify the precise isomeric form. To advance the techniques available for glycan analysis, we employ two important innovations. First, we perform ultrahigh-resolution mobility separation using structures for lossless ion manipulations (SLIM) for isomer separation and pre-selection. We then complement this IMS-MS stage with a cryogenic IR spectroscopic dimension since a glycan's vibrational spectrum provides a fingerprint that is extremely sensitive to the precise isomeric form. Using this unique approach in conjunction with oxygen-18 isotopic labeling, we show on a range of disaccharides how the two α and β anomers that every reducing glycan adopts in solution can be readily separated by mobility and identified based on their IR spectra. In addition to highlighting the power of our technique to detect minute differences in the structure of isomeric carbohydrates, these results provide the means to determine if and when anomericity is retained during collision-induced dissociation (CID) of larger glycans.

SLIM Ultrahigh Resolution Ion Mobility Spectrometry Separations of Isotopologues and Isotopomers Reveal Mobility Shifts due to Mass Distribution Changes.

We report on separations of ion isotopologues and isotopomers using ultrahigh-resolution traveling wave-based Structures for Lossless Ion Manipulations with serpentine ultralong path and extended routing ion mobility spectrometry coupled to mass spectrometry (SLIM SUPER IMS-MS). Mobility separations of ions from the naturally occurring ion isotopic envelopes (e.g., [M], [M+1], [M+2], ... ions) showed the first and second isotopic peaks (i.e., [M+1] and [M+2]) for various tetraalkylammonium ions could be resolved from their respective monoisotopic ion peak ([M]) after SLIM SUPER IMS with resolving powers of ∼400-600. Similar separations were obtained for other compounds (e.g., tetrapeptide ions). Greater separation was obtained using argon versus helium drift gas, as expected from the greater reduced mass contribution to ion mobility described by the Mason-Schamp relationship. To more directly explore the role of isotopic substitutions, we studied a mixture of specific isotopically substituted (15N, 13C, and 2H) protonated arginine isotopologues. While the separations in nitrogen were primarily due to their reduced mass differences, similar to the naturally occurring isotopologues, their separations in helium, where higher resolving powers could also be achieved, revealed distinct additional relative mobility shifts. These shifts appeared correlated, after correction for the reduced mass contribution, with changes in the ion center of mass due to the different locations of heavy atom substitutions. The origin of these apparent mass distribution-induced mobility shifts was then further explored using a mixture of Iodoacetyl Tandem Mass Tag (iodoTMT) isotopomers (i.e., each having the same exact mass, but with different isotopic substitution sites). Again, the observed mobility shifts appeared correlated with changes in the ion center of mass leading to multiple monoisotopic mobilities being observed for some isotopomers (up to a ∼0.04% difference in mobility). These mobility shifts thus appear to reflect details of the ion structure, derived from the changes due to ion rotation impacting collision frequency or momentum transfer, and highlight the potential for new approaches for ion structural characterization.

Combining ultra-high resolution ion mobility spectrometry with cryogenic IR spectroscopy for the study of biomolecular ions.

Double-resonance spectroscopic schemes in combination with cryogenic ion traps are the go-to techniques when isomer-specific high-resolution spectra are required for analysis of molecular ions. Their limitation lies in the requirement for well-resolved, isomer-specific absorption bands as well as in the potentially time-consuming steps to identify each isomer. We present an alternative approach where isomeric species are readily separated using ion mobility spectrometry (IMS) and selected prior to cryogenic spectroscopic analysis. To date, most IMS approaches suffer from relatively low resolution, however, recent technological developments in the field of travelling-wave ion mobility using structures for lossless ion manipulation (SLIM) permit the use of extremely long drift paths, which greatly enhances the resolution. We demonstrate the power of combining this type of ultra-high resolution IMS with cryogenic vibrational spectroscopy by comparing mobility-resolved IR spectra of a disaccharide to those acquired using IR-IR double resonance. This new approach is especially promising for the investigation of larger molecules where spectral congestion interferes with double resonance techniques.

Lipid and Glycolipid Isomer Analyses Using Ultra-High Resolution Ion Mobility Spectrometry Separations.

Understanding the biological roles and mechanisms of lipids and glycolipids is challenging due to the vast number of possible isomers that may exist. Mass spectrometry (MS) measurements are currently the dominant approach for studying and providing detailed information on lipid and glycolipid presence and changes. However, difficulties in distinguishing the many structural isomers, due to the distinct lipid acyl chain positions, double bond locations or specific glycan types, inhibit the delineation and assignment of their biological roles. Here we utilized ultra-high resolution ion mobility spectrometry (IMS) separations by applying traveling waves in a serpentine multi-pass Structures for Lossless Ion Manipulations (SLIM) platform to enhance the separation of selected lipid and glycolipid isomers. The multi-pass arrangement allowed the investigation of paths ranging from ~16 m (one pass) to ~60 m (four passes) for the distinction of lipids and glycolipids with extremely small structural differences. These ultra-high resolution SLIM IMS-MS analyses provide a foundation for exploring and better understanding isomer-specific biological activities and disease processes.

Distinguishing d- and l-aspartic and isoaspartic acids in amyloid β peptides with ultrahigh resolution ion mobility spectrometry.

While α-linked amino acids in the l-form are exclusively utilized in mammalian protein building, β-linked and d-form amino acids also have important biological roles. Unfortunately, the structural elucidation and separation of these different amino acid types in peptides has been analytically challenging to date due to the numerous isomers present, limiting our knowledge about their existence and biological roles. Here, we utilized an ultrahigh resolution ion mobility spectrometry platform coupled with mass spectrometry (IMS-MS) to separate amyloid β (Aβ) peptides containing l-aspartic acid, d-aspartic acid, l-isoaspartic acid, and d-isoaspartic acid residues which span α- and β-linked amino acids in both d- and l-forms. The results illustrate how IMS-MS could be used to better understand age-related diseases or protein folding disorders resulting from amino acid modifications.

Evaluation of ultrahigh resolution ion mobility spectrometry as an analytical separation device in chromatographic terms

An ion mobility spectrometer (IMS) that routinely achieves separation efficiencies in line with the theoretical diffusion limited maximum has been constructed. The theoretical maximum efficiency is a function of the total voltage drop, the temperature, the number of charges on the ion, the initial pulse width, the length of the spectrometer, and the mobility (K) of the ion of interest. Despite the fact that our current instrumental setup uses modest voltages and fairly high temperatures, we were able to achieve over 130,000 theoretical plates for a singly charged ion in less than 25 ms. In addition, selectivity can be altered in IMS by changing the drift gas or by increasing the electric field (>1000 V/cm) such that the mobility of ions is no longer a linear function of the electric field. Separation factors (α) can be altered by as much as 20% by changing the drift gas in the spectrometer. Finally leucine and isoleucine were separated in less than 23 ms with a resolution of 0.668 demonstrating the separation power of the instrument. © 2000 John Wiley & Sons, Inc. J Micro Sep 12: 172–178, 2000

Evaluation of ultrahigh resolution ion mobility spectrometry as an analytical separation device in chromatographic terms * Presented at the Twenty-first International Symposium on Capillary Chromatography and Electrophoresis, Park City, Utah, USA, June 20–24, 1999.

Evaluation of ultrahigh resolution ion mobility spectrometry as an analytical separation device in chromatographic terms * Presented at the Twenty-first International Symposium on Capillary Chromatography and Electrophoresis, Park City, Utah, USA, June 20–24, 1999.