Leveraging High-Resolution Ion Mobility-Mass Spectrometry for Cyclic Peptide Soft Spot Identification.

Cyclic peptides are an important class of molecules that gained significant attention in the field of drug discovery due to their unique pharmacological characteristics and enhanced proteolytic stability. Yet, gastrointestinal degradation remains a major hurdle in the discovery of orally bioavailable cyclic peptides. Soft spot identification (SSID) of the regions in the cyclic peptide sequence susceptible to amide hydrolysis by proteases is used in the discovery stage to guide medicinal chemistry design. SSID can be an arduous task, traditionally performed using liquid chromatography-tandem mass spectrometry (LC-MS/MS), often resulting in complex and time-consuming manual analysis, particularly when isomeric linear peptide metabolites chromatographically coelute. Here, we present an alternative orthogonal approach that entails a high-resolution ion mobility (HRIM) system based on Structures for Lossless Ion Manipulation (SLIM) technology interfaced with quadrupole time-of-flight (QTOF) mass spectrometry to address some of the challenges associated with SSID. Two strategies were used to resolve linear isomeric peptide metabolites: labeled and label-free, both utilizing the HRIM platform. The label-free strategy leverages negative polarity to ionize the isomers which achieves better separation of the gas phase ions in the ion mobility (IM) dimension as compared to positive polarity, which is a more conventional approach when studying proteins and peptides. The second approach uses an isotope-labeled dimethyl tag on the terminal amine group, acting as a "shift reagent" to influence the mobility of isomers in the positive mode. This method resulted in baseline separation for the isomers of interest and produced unique product ions in the fragmentation spectra for unambiguous soft spot identification. Both label-free and labeled strategies demonstrated the ability to solve the challenges associated with SSID for cyclic peptides.

The Future of Proteomics is Up in the Air: Can Ion Mobility Replace Liquid Chromatography for High Throughput Proteomics?

The coevolution of liquid chromatography (LC) with mass spectrometry (MS) has shaped contemporary proteomics. LC hyphenated to MS now enables quantification of more than 10,000 proteins in a single injection, a number that likely represents most proteins in specific human cells or tissues. Separations by ion mobility spectrometry (IMS) have recently emerged to complement LC and further improve the depth of proteomics. Given the theoretical advantages in speed and robustness of IMS in comparison to LC, we envision that ongoing improvements to IMS paired with MS may eventually make LC obsolete, especially when combined with targeted or simplified analyses, such as rapid clinical proteomics analysis of defined biomarker panels. In this perspective, we describe the need for faster analysis that might drive this transition, the current state of direct infusion proteomics, and discuss some technical challenges that must be overcome to fully complete the transition to entirely gas phase proteomics.

Investigating Performance of the SLIM-Based High Resolution Ion Mobility Platform for Separation of Isomeric Phosphatidylcholine Species.

Lipids are structurally diverse molecules that play a pivotal role in a plethora of biological processes. However, deciphering the biological roles of the specific lipids is challenging due to the existence of numerous isomers. This high chemical complexity of the lipidome is one of the major challenges in lipidomics research, as the traditional liquid chromatography-mass spectrometry (LC-MS) based approaches are often not powerful enough to resolve these isomeric and isobaric nuances within complex samples. Thus, lipids are uniquely suited to the benefits provided by multidimensional liquid chromatography-ion mobility-mass spectrometry (LC-IM-MS) analysis. However, many forms of lipid isomerism, including double-bond positional isomers and regioisomers, are structurally similar such that their collision cross section (CCS) differences are unresolvable via conventional IM approaches. Here we evaluate the performance of a high resolution ion mobility (HRIM) system based on structures for lossless ion manipulation (SLIM) technology interfaced to a high resolution quadrupole time-of-flight (QTOF) analyzer to address the noted lipidomic isomerism challenge. SLIM implements the traveling wave ion mobility technique along an ∼13 m ion path, providing longer path lengths to enable improved separation of isomeric features. We demonstrate the power of HRIM-MS to dissect isomeric PC standards differing only in double bond (DB) and stereospecific number (SN) positions. The partial separation of protonated DB isomers is significantly enhanced when they are analyzed as metal adducts. For sodium adducts, we achieve close to baseline separation of three different PC 18:1/18:1 isomers with different cis-double bond locations. Similarly, PC 18:1/18:1 (cis-9) can be resolved from the corresponding PC 18:1/18:1 (trans-9) form. The separation capacity is further enhanced when using silver ion doping, enabling the baseline separation of regioisomers that cannot be resolved when measured as sodium adducts. The sensitivity and reproducibility of the approach were assessed, and the performance for more complex mixtures was benchmarked by identifying PC isomers in total brain and liver lipid extracts.

High-Resolution Ion-Mobility-Enabled Peptide Mapping for High-Throughput Critical Quality Attribute Monitoring.

Characterization and monitoring of post-translational modifications (PTMs) by peptide mapping is a ubiquitous assay in biopharmaceutical characterization. Often, this assay is coupled to reversed-phase liquid chromatographic (LC) separations that require long gradients to identify all components of the protein digest and resolve critical modifications for relative quantitation. Incorporating ion mobility (IM) as an orthogonal separation that relies on peptide structure can supplement the LC separation by providing an additional differentiation filter to resolve isobaric peptides, potentially reducing ambiguity in identification through mobility-aligned fragmentation and helping to reduce the run time of peptide mapping assays. A next-generation high-resolution ion mobility (HRIM) technique, based on structures for lossless ion manipulations (SLIM) technology with a 13 m ion path, provides peak capacities and higher resolving power that rivals traditional chromatographic separations and, owing to its ability to resolve isobaric peptides that coelute in faster chromatographic methods, allows for up to 3× shorter run times than conventional peptide mapping methods. In this study, the NIST monoclonal antibody IgG1κ (NIST RM 8671, NISTmAb) was characterized by LC-HRIM-MS and LC-HRIM-MS with collision-induced dissociation (HRIM-CID-MS) using a 20 min analytical method. This approach delivered a sequence coverage of 96.5%. LC-HRIM-CID-MS experiments provided additional confidence in sequence determination. HRIM-MS resolved critical oxidations, deamidations, and isomerizations that coelute with their native counterparts in the chromatographic dimension. Finally, quantitative measurements of % modification were made using only the m/z-extracted HRIM arrival time distributions, showing good agreement with the reference liquid-phase separation. This study shows, for the first time, the analytical capability of HRIM using SLIM technology for enhancing peptide mapping workflows relevant to biopharmaceutical characterization.

Resolving Power and Collision Cross Section Measurement Accuracy of a Prototype High-Resolution Ion Mobility Platform Incorporating Structures for Lossless Ion Manipulation.

A production prototype structures for lossless ion manipulation ion mobility (SLIM IM) platform interfaced to a commercial high-resolution mass spectrometer (MS) is described. The SLIM IM implements the traveling wave ion mobility technique across a ∼13m path length for high-resolution IM (HRIM) separations. The resolving power (CCS/ΔCCS) of the SLIM IM stage was benchmarked across various parameters (traveling wave speeds, amplitudes, and waveforms), and results indicated that resolving powers in excess of 200 can be accessed for a broad range of masses. For several cases, resolving powers greater than 300 were achieved, notably under wave conditions where ions transition from a nonselective "surfing" motion to a mobility-selective ion drift, that corresponded to ion speeds approximately 30-70% of the traveling wave speed. The separation capabilities were evaluated on a series of isomeric and isobaric compounds that cannot be resolved by MS alone, including reversed-sequence peptides (SDGRG and GRGDS), triglyceride double-bond positional isomers (TG 3, 6, 9 and TG 6, 9, 12), trisaccharides (melezitose, raffinose, isomaltotriose, and maltotriose), and ganglioside lipids (GD1b and GD1a). The SLIM IM platform resolved the corresponding isomeric mixtures, which were unresolvable using the standard resolution of a drift-tube instrument (∼50). In general, the SLIM IM-MS platform is capable of resolving peaks separated by as little as ∼0.6% without the need to target a specific separation window or drift time. Low CCS measurement biases <0.5% were obtained under high resolving power conditions. Importantly, all the analytes surveyed are able to access high-resolution conditions (>200), demonstrating that this instrument is well-suited for broadband HRIM separations important in global untargeted applications.

High-defined quantitative snapshots of the ganglioside lipidome using high resolution ion mobility SLIM assisted shotgun lipidomics.

Defects in sphingolipid metabolism have emerged as a common link across neurodegenerative disorders, and a deeper understanding of the lipid content in preclinical models and patient specimens offers opportunities for development of new therapeutic targets and biomarkers. Sphingolipid metabolic pathways include the formation of glycosphingolipid species that branch into staggeringly complex structural heterogeneity within the globoside and ganglioside sub-lipidomes. Characterization of these sub-lipidomes has typically relied on liquid chromatography-mass spectrometry-based (LC-MS) approaches, but such assays are challenging and resource intensive due to the close structural heterogeneity, the presence of isobaric and isomeric species, and broad dynamic range of endogenous glycosphingolipids. Here, we apply Structures for Lossless Ion Manipulations (SLIM)-based High Resolution Ion Mobility (HRIM)-MS to enable rapid, repeatable, quantitative assays with deep structural information sufficient to resolve endogenous brain gangliosides at the level of individual molecular species. Analyses were performed using a prototype SLIM-MS instrument equipped with a 13-m serpentine path which enabled resolution of closely related isomeric analytes such as GD1a d36:1 and GD1b d36:1 based on recorded mass-to-charge (m/z) and arrival times. To demonstrate the power of our methodology, brain extracts derived from wild-type mice hemi-brains were analyzed by HRIM-MS using flow injection analyses (FIA) without the need for additional separation by liquid chromatography. Endogenous ganglioside species were readily resolved, identified, and quantified by FIA-SLIM-MS analyses within 2 min per sample. Thus, the FIA-SLIM-MS platform enables robust quantification across a broad range of lipid species in biological specimens in a standardized assay format that is readily scalable to support studies with large sample numbers.

The Potential for Ion Mobility in Pharmaceutical and Clinical Analyses.

The pharmaceutical and clinical industries are imperative for the maintenance of global health and welfare and require accurate, reproducible, and high throughput analyses. Technological advancements, such as the development and implementation of liquid chromatography-tandem mass spectrometry (LC-MS), have allowed for improvements in these areas, however there is still room for development. One way in which current analyses may be improved is by the implementation of ion mobility technology. Ion mobility has the capability to produce much more comprehensive data sets, by providing separation of isomers, as well as improving throughput, with separations being performed as fast as 60 ms. Here we will discuss the potential for ion mobility to assist in the two specific areas of glycosylation monitoring of biological drugs, and vitamin D analysis, as representatives of ion mobility's potential in both the pharmaceutical and clinical industries, respectively, as well as the current hurdles of ion mobility adoption in both fields.