Exploring Ion Mobility Mass Spectrometry Data File Conversions to Leverage Existing Tools and Enable New Workflows.

Ion mobility (IM) is often combined with LC-MS experiments to provide an additional dimension of separation for complex sample analysis. While highly complex samples are better characterized by the full dimensionality of LC-IM-MS experiments to uncover new information, downstream data analysis workflows are often not equipped to properly mine the additional IM dimension. For many samples the data acquisition benefits of including IM separations are all that is necessary to uncover sample information and the full dimensionality of the data is not required for data analysis.

CIUSuite 3: Next-Generation CCS Calibration and Automated Data Analysis Tools for Gas-Phase Protein Unfolding Data.

Ion mobility-mass spectrometry (IM-MS) has become a technology deployed across a wide range of structural biology applications despite the challenges in characterizing closely related protein structures. Collision-induced unfolding (CIU) has emerged as a valuable technique for distinguishing closely related, iso-cross-sectional protein and protein complex ions through their distinct unfolding pathways in the gas phase. With the speed and sensitivity of CIU analyses, there has been a rapid growth of CIU-based assays, especially regarding biomolecular targets that remain challenging to assess and characterize with other structural biology tools.

Effects of Nano-Electrospray Ionization Emitter Position on Unintentional In-Source Activation of Peptide and Protein Ions.

Native ion mobility-mass spectrometry (IM-MS) typically introduces protein ions into the gas phase through nano-electrospray ionization (nESI). Many nESI setups have mobile stages for tuning the ion signal and extent of co-solute and salt adduction. However, tuning the position of the emitter capillary in nESI can have unintended downstream consequences for collision-induced unfolding or collision-induced dissociation (CIU/D) experiments.

Performance evaluation of in-source ion activation hardware for collision-induced unfolding of proteins and protein complexes on a drift tube ion mobility-mass spectrometer.

Native ion mobility-mass spectrometry (IM-MS) has emerged as an information-rich technique for gas phase protein structure characterization; however, IM resolution is currently insufficient for the detection of subtle structural differences in large biomolecules. This challenge has spurred the development of collision-induced unfolding (CIU) which utilizes incremental gas phase activation to unfold a protein in order to expand the number of measurable descriptors available for native protein ions. Although CIU is now routinely used in native mass spectrometry studies, the interlaboratory reproducibility of CIU has not been established.

Collision-Induced Unfolding Reveals Stability Differences in Infliximab Therapeutics under Native and Heat Stress Conditions.

Ion mobility-mass spectrometry (IM-MS) and collision-induced unfolding (CIU) assays of monoclonal antibody (mAb)-based biotherapeutics have proven sensitive to disulfide bridge structures, glycosylation patterns, and small molecule conjugation levels. Despite promising prior reports detailing the capabilities of IM-MS and CIU to differentiate biosimilars, generic mAb therapeutics, there remain questions surrounding the sensitivity of CIU to mAb structure changes that occur upon stress, the reproducibility of such measurements across IM-MS platforms, and the correlation between CIU and differential scanning calorimetry (DSC) datasets. In this report, we describe a comprehensive IM-MS and CIU dataset acquired for three Infliximabs: Remicade, Inflectra, and Renflexis.

Novel acquisition strategies for metabolomics using drift tube ion mobility-quadrupole resolved all ions time-of-flight mass spectrometry (IM-QRAI-TOFMS).

The focus of this work was the implementation of ion mobility (IM) and a prototype quadrupole driver within data independent acquisition (DIA) using a drift tube IM-QTOFMS aiming to improve the level of confidence in identity confirmation workflows for non-targeted metabolomics. In addition to conventional all ions (IM-AI) acquisition, quadrupole resolved all ions (IM-QRAI) acquisition allows a drift time-directed precursor ion isolation in DIA using sequential isolation of precursor ions using mass windows of up to 100 Da which can be rapidly ramped across single ion mobility transients (i.e.

Enhanced Collision Induced Unfolding and Electron Capture Dissociation of Native-like Protein Ions.

Native ion mobility-mass spectrometry (IM-MS) is capable of revealing much that remains unknown within the structural proteome, promising such information on refractory protein targets. Here, we report the development of a unique drift tube IM-MS (DTIM-MS) platform, which combines high-energy source optics for improved collision induced unfolding (CIU) experiments and an electromagnetostatic cell for electron capture dissociation (ECD). We measured a series of high precision collision cross section (CCS) values for protein and protein complex ions ranging from 6-1600 kDa, exhibiting an average relative standard deviation (RSD) of 0.

Ion Mobility-Mass Spectrometry Imaging Workflow.

Mass spectrometry imaging (MSI) is a powerful technique for the label-free spatially resolved analysis of biological tissues. Coupling ion mobility (IM) separation with MSI allows for separation of isobars in the mobility dimension and increases confidence of peak assignments. Recently, imaging experiments have been implemented on several commercially available and custom-designed ion mobility instruments, making IM-MSI experiments more broadly accessible to the MS community.

Collision-Induced Unfolding Studies of Proteins and Protein Complexes using Drift Tube Ion Mobility-Mass Spectrometer.

Elucidating the structures and stabilities of proteins and their complexes is paramount to understanding their biological functions in cellular processes. Native mass spectrometry (MS) coupled with ion mobility spectrometry (IMS) is emerging as an important biophysical technique owing to its high sensitivity, rapid analysis time, and ability to interrogate sample complexity or heterogeneity and the ability to probe protein structure dynamics. Here, a commercial IMS-MS platform has been modified for static native ESI emitters and an extended mass-to-charge range (20 kDa /) and its performance capabilities and limits were explored for a range of protein and protein complexes.

Drift-Tube Ion Mobility-Mass Spectrometry for Nontargeted 'Omics.

This chapter describes the developments in drift-tube ion mobility-mass spectrometry (DTIM-MS) that have driven application development in 'omics analyses. Harnessing the additional, orthogonal separation that DTIM provides increased confidence in compound identifications as the mass spectral complexity can be reduced and mobility-derived parameters (most prominently the collision cross section, CCS) used to support identity confirmation goals for a variety of 'omics application areas. Presented within this contribution is a methodology for improving the transmission and maintaining accurate determination of drift time-derived CCS (CCS) for low molecular weight compounds for a typical nontargeted 'omics (metabolomics) workflow using liquid chromatography in combination with DTIM-MS.

Fundamental study of ion trapping and multiplexing using drift tube-ion mobility time-of-flight mass spectrometry for non-targeted metabolomics.

This study of ion accumulation/release behavior relevant to ion mobility-mass spectrometry (IM-MS) as employed for non-targeted metabolomics involves insight from theoretical studies, and controlled reference experiments involving measurement of low and high molecular mass metabolites in varying concentrations within a complex matrix (yeast extracts). Instrumental settings influencing ion trapping (accumulation time) and release conditions in standard and multiplexed operation have been examined, and translation of these insights to liquid chromatography (LC) in combination with drift tube IM-MS measurements has been made. The focus of the application is non-targeted metabolomics using carefully selected samples to allow quantitative interpretations to be made.

A Modified Drift Tube Ion Mobility-Mass Spectrometer for Charge-Multiplexed Collision-Induced Unfolding.

Collision-induced unfolding (CIU) of protein ions and their noncovalent complexes offers relatively rapid access to a rich portfolio of biophysical information, without the need to tag or purify proteins prior to analysis. Such assays have been characterized extensively for a range of therapeutic proteins, proving exquisitely sensitive to alterations in protein sequence, structure, and post-translational modification state. Despite advantages over traditional probes of protein stability, improving the throughput and information content of gas-phase protein unfolding assays remains a challenge for current instrument platforms.

Rapid screening methods for yeast sub-metabolome analysis with a high-resolution ion mobility quadrupole time-of-flight mass spectrometer.

Rationale: The wide chemical diversity and complex matrices inherent to metabolomics still pose a challenge to current analytical approaches for metabolite screening. Although dedicated front-end separation techniques combined with high-resolution mass spectrometry set the benchmark from an analytical point of view, the increasing number of samples and sample complexity demand for a compromise in terms of selectivity, sensitivity and high-throughput analyses.

Methods: Prior to low-field drift tube ion mobility (IM) separation and quadrupole time-of-flight mass spectrometry (QTOFMS) detection, rapid ultrahigh-performance liquid chromatography separation was used for analysis of different concentration levels of dansylated metabolites present in a yeast cell extract.

Recommendations for reporting ion mobility Mass Spectrometry measurements.

Here we present a guide to ion mobility mass spectrometry experiments, which covers both linear and nonlinear methods: what is measured, how the measurements are done, and how to report the results, including the uncertainties of mobility and collision cross section values. The guide aims to clarify some possibly confusing concepts, and the reporting recommendations should help researchers, authors and reviewers to contribute comprehensive reports, so that the ion mobility data can be reused more confidently. Starting from the concept of the definition of the measurand, we emphasize that (i) mobility values (K ) depend intrinsically on ion structure, the nature of the bath gas, temperature, and E/N; (ii) ion mobility does not measure molecular surfaces directly, but collision cross section (CCS) values are derived from mobility values using a physical model; (iii) methods relying on calibration are empirical (and thus may provide method-dependent results) only if the gas nature, temperature or E/N cannot match those of the primary method.

Cyclodextrin and malto-dextrose collision cross sections determined in a drift tube ion mobility mass spectrometer using nitrogen bath gas.

In this study, we have evaluated a low field limit drift tube ion mobility device for ion mobility-mass spectrometry (IM-MS) measurements that uses nitrogen as a bath gas with electrospray ionization on a modified Q-TOF instrument. We have determined reduced mobility (K0) and collision cross section (CCS) values for a group of analyte ions that have been characterized previously in other drift tube IM-MS instruments. Our determinations of CCS for this set of ions as well as for standards are in agreement with published values.

SPE-IMS-MS: An automated platform for sub-sixty second surveillance of endogenous metabolites and xenobiotics in biofluids.

Characterization of endogenous metabolites and xenobiotics is essential to deconvoluting the genetic and environmental causes of disease. However, surveillance of chemical exposure and disease-related changes in large cohorts requires an analytical platform that offers rapid measurement, high sensitivity, efficient separation, broad dynamic range, and application to an expansive chemical space. Here, we present a novel platform for small molecule analyses that addresses these requirements by combining solid-phase extraction with ion mobility spectrometry and mass spectrometry (SPE-IMS-MS).

An Interlaboratory Evaluation of Drift Tube Ion Mobility-Mass Spectrometry Collision Cross Section Measurements.

Collision cross section (CCS) measurements resulting from ion mobility-mass spectrometry (IM-MS) experiments provide a promising orthogonal dimension of structural information in MS-based analytical separations. As with any molecular identifier, interlaboratory standardization must precede broad range integration into analytical workflows. In this study, we present a reference drift tube ion mobility mass spectrometer (DTIM-MS) where improvements on the measurement accuracy of experimental parameters influencing IM separations provide standardized drift tube, nitrogen CCS values (CCS) for over 120 unique ion species with the lowest measurement uncertainty to date.

Broadscale resolving power performance of a high precision uniform field ion mobility-mass spectrometer.

An extensive study of two current ion mobility resolving power theories ("conditional" and "semi-empirical") was undertaken using a recently developed drift tube ion mobility-mass spectrometer. The current study investigates the quantitative agreement between experiment and theory at reduced pressure (4 Torr) for a wide range of initial ion gate widths (100 to 500 μs), and ion mobility values (K0 from 0.50 to 3.

Evaluation of drift gas selection in complex sample analyses using a high performance drift tube ion mobility-QTOF mass spectrometer.

A recently developed uniform-field high resolution ion mobility (IM) quadrupole time of flight (Q-TOF) mass spectrometer is used for evaluating the utility of alternate drift gases for complex sample analyses. This study provides collision cross section comparison for 275 total pesticides including structural isomers in nitrogen, helium, carbon dioxide, nitrous oxide and sulfur hexafluoride drift gases. Furthermore, a set of small molecules and Agilent tune mix compounds were used to study the trends in experimentally derived collision cross section values in argon and the alternate drift gases.

Conformational ordering of biomolecules in the gas phase: nitrogen collision cross sections measured on a prototype high resolution drift tube ion mobility-mass spectrometer.

Ion mobility-mass spectrometry measurements which describe the gas-phase scaling of molecular size and mass are of both fundamental and pragmatic utility. Fundamentally, such measurements expand our understanding of intrinsic intramolecular folding forces in the absence of solvent. Practically, reproducible transport properties, such as gas-phase collision cross-section (CCS), are analytically useful metrics for identification and characterization purposes.

Delineating diseases by IMS-MS profiling of serum N-linked glycans.

Altered branching and aberrant expression of N-linked glycans is known to be associated with disease states such as cancer. However, the complexity of determining such variations hinders the development of specific glycomic approaches for assessing disease states. Here, we examine a combination of ion mobility spectrometry (IMS) and mass spectrometry (MS) measurements, with principal component analysis (PCA) for characterizing serum N-linked glycans from 81 individuals: 28 with cirrhosis of the liver, 25 with liver cancer, and 28 apparently healthy.

Overtone mobility spectrometry: part 4. OMS-OMS analyses of complex mixtures.

A new, two-dimensional overtone mobility spectrometry (OMS-OMS) instrument is described for the analysis of complex peptide mixtures. OMS separations are based on the differences in mobilities of ions in the gas phase. The method utilizes multiple drift regions with modulated drift fields such that only ions with appropriate mobilities are transmitted to the detector.

Overtone mobility spectrometry: part 3. On the origin of peaks.

The origin of non-integer overtone peaks in overtone mobility spectrometry (OMS) spectra is investigated by ion trajectory simulations. Simulations indicate that these OMS features arise from higher-order overtone series. An empirically-derived formula is presented as a means of describing the positions of peaks.

Determination of cross sections by overtone mobility spectrometry: evidence for loss of unstable structures at higher overtones.

Overtone mobility spectrometry (OMS) is examined as a means of determining the collision cross sections for multiply charged ubiquitin and substance P ions, as well as for singly charged rafinose and melezitose ions. Overall, values of collision cross section measured by OMS for stable ion conformations are found to be in agreement with values determined by conventional ion mobility spectrometry (IMS) measurements to within ∼1% relative uncertainty. The OMS spectra for ubiquitin ions appear to favor different conformations at higher overtones.

Overtone mobility spectrometry: part 2. Theoretical considerations of resolving power.

The transport of ions through multiple drift regions is modeled to develop an equation that is useful for an understanding of the resolving power of an overtone mobility spectrometry (OMS) technique. It is found that resolving power is influenced by a number of experimental variables, including those that define ion mobility spectrometry (IMS) resolving power: drift field (E), drift region length (L), and buffer gas temperature (T). However, unlike IMS, the resolving power of OMS is also influenced by the number of drift regions (n), harmonic frequency value (m), and the phase number (Phi) of the applied drift field.