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O-acetylation is a common modification of sialic acids that has been implicated in a multitude of biological and disease processes. A lack of analytical methods that can determine exact structures of sialic acid variants is a hurdle to determine roles of distinct O-acetylated sialosides. Here, we describe a drift tube ion mobility-mass spectrometry approach that can elucidate exact O-acetylation patterns as well as glycosidic linkage types of sialosides isolated from complex biological samples. It is based on the use of a library of synthetic O-acetylated sialosides to establish intrinsic collision cross section (CCS) values of diagnostic fragment ions. The CCS values were used to characterize O-acetylated sialosides from mucins and N-linked glycans from biologicals as well as equine tracheal and nasal tissues. It uncovered contrasting sialic acid linkage types of acetylated and non-acetylated sialic acids and provided a rationale for sialic acid binding preferences of equine H7 influenza A viruses., (© 2023. Springer Nature Limited.)

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.

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. However, the absence of open access data analysis software for IM-MSI systems presents a bottleneck. Herein, we present an imaging workflow to visualize IM-MSI data produced on the Agilent 6560 ion mobility quadrupole time-of-flight system. Specifically, we have developed a Python script, the ion mobility-mass spectrometry image creation script (IM-MSIC), which interfaces Agilent's Mass Hunter Mass Profiler software with the MacCoss lab's Skyline software and generates drift time and mass-to-charge-selected ion images. In the workflow, Mass Profiler is used for an untargeted feature detection. The IM-MSIC script mediates user input of data, extracts ion chronograms utilizing Skyline's command-line interface, and then proceeds toward ion image generation within a single user interface. Ion image postprocessing is subsequently performed using different tools implemented in accompanying scripts. Though the current work only showcases Agilent IM-MSI data, this workflow can be readily adapted for use with most major instrument vendors.

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. Experimental investigation of the IM-MS ion utilization efficiency particularly focusing on the use of the Hadamard transform multiplexing with 4-bit pseudo-random pulsing sequence for assessment of low and high molecular mass metabolites is compared with theoretical modeling of gas-phase behavior of small and large molecules in the IM trapping funnel. Increasing the trapping time for small metabolites with standard IM-MS operation is demonstrated to have a deleterious effect on maintaining a quantitative representation of the metabolite abundance. The application of these insights to real-world non-targeted metabolomics assessment of intracellular extracts from biotechnologically relevant production processes is presented, and the results were compared to LC×IM-MS measurements of the same samples. Spiking of a uniformly 13 C-labeled yeast extract (as a standard matrix) with varying amounts of natural metabolites is used to assess the linearity and sensitivity according to the instrument mode of operation (i.e., LC-MS, LC×IM-MS, and LC×[multiplexed]IM-MS). When comparing metabolite quantification using standard and multiplexed operation, sensitivity gain factors of 2-8 were obtained for metabolites with m/z below 250. Taken together, the simulation and experimental results of this study provide insight for optimizing measurement conditions for metabolomics and highlight the need for implementation of multiplexing strategies using short trapping times as relative quantification (e.g., in the context with non-targeted differential analysis) with sufficient sensitivity and working range is a requirement in this field of application.

Analysis of oxylipins by liquid chromatography mass spectrometry (LC/MS) is challenging because of the small mass range occupied by this diverse lipid class, the presence of numerous structural isomers, and their low abundance in biological samples. Although highly sensitive LC/MS/MS methods are commonly used, further separation is achievable by using drift tube ion mobility coupled with high-resolution mass spectrometry (DTIM-MS). Herein, we present a combined analytical and computational method for the identification of oxylipins and fatty acids. We use a reversed-phase LC/DTIM-MS workflow able to profile and quantify (based on chromatographic peak area) the oxylipin and fatty acid content of biological samples while simultaneously acquiring full scan and product ion spectra. The information regarding accurate mass, collision-cross-section values in nitrogen ( DT CCS N2 ), and retention times of the species found are compared to an internal library of lipid standards as well as the LIPID MAPS Structure Database by using specifically developed processing tools. Features detected within the DT CCS N2 and m/ z ranges of the analyzed standards are flagged as oxylipin-like species, which can be further characterized using drift-time alignment of product and precursor ions distinctive of DTIM-MS. This not only helps identification by reducing the number of annotations from LIPID MAPS but also guides discovery studies of potentially novel species. Testing the methodology on Salmonella enterica serovar Typhimurium-infected murine bone-marrow-derived macrophages and thrombin activated human platelets yields results in agreement with literature. This workflow has also annotated features as potentially novel oxylipins, confirming its ability in providing further insights into lipid analysis of biological samples.

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. For identity confirmation of metabolites at the MS2 level, an alternating frame approach was chosen and two different strategies were tested: a data-independent all-ions acquisition and a quadrupole broad band isolation (Q-BBI) directed by IM drift separation., Results: For Q-BBI analysis, the broad mass range isolation was successfully optimized in accordance with the distinctive drift time to m/z correlation of the dansyl derivatives. To guarantee comprehensive sampling, a broad mass isolation window of 70 Da was employed. Fragmentation was performed via collision-induced dissociation, applying a collision energy ramp optimized for the dansyl derivatives. Both approaches were studied in terms of linear dynamic range and repeatability employing ethanolic extracts of Pichia pastoris spiked with 1 μM metabolite mixture. Example data obtained for histidine and glycine showed that drift time precision (<0.01 to 0.3% RSD, n = 5) compared very well with the data reported in an earlier IM-TOFMS-based study., Conclusions: Chimeric mass spectra, inherent to data-independent analysis approaches, are reduced when using a drift time directed Q-BBI approach. Additionally, an improved linear dynamic working range was observed, representing, together with a rapid front-end separation, a powerful approach for metabolite screening., (© 2019 The Authors Rapid Communications in Mass Spectrometry Published by John Wiley & Sons, Ltd.)

Introduction: Data processing is one of the biggest problems in metabolomics, given the high number of samples analyzed and the need of multiple software packages for each step of the processing workflow., Objectives: Merge in the same platform the steps required for metabolomics data processing., Methods: KniMet is a workflow for the processing of mass spectrometry-metabolomics data based on the KNIME Analytics platform., Results: The approach includes key steps to follow in metabolomics data processing: feature filtering, missing value imputation, normalization, batch correction and annotation., Conclusion: KniMet provides the user with a local, modular and customizable workflow for the processing of both GC-MS and LC-MS open profiling data., Competing Interests: Compliance with ethical standardsAuthors declare that they have no conflict of interest.This article does not contain any studies with human participants or animals performed by any of the authors.

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). This platform is capable of performing both targeted and global measurements of endogenous metabolites and xenobiotics in human biofluids with high reproducibility (CV 6 3%), sensitivity (LODs in the pM range in biofluids) and throughput (10-s sample-to-sample duty cycle). We report application of this platform to the analysis of human urine from patients with and without type 1 diabetes, where we observed statistically significant variations in the concentration of disaccharides and previously unreported chemical isomers. This SPE-IMS-MS platform overcomes many of the current challenges of large-scale metabolomic and exposomic analyses and offers a viable option for population and patient cohort screening in an effort to gain insights into disease processes and human environmental chemical exposure., Competing Interests: Competing financial interests The co-authors, Y.M.I and R.D.S., are co-inventors on one of the patents licensed by Agilent for the 6560 IMS-QTOF MS instrument utilized in this work. M.R., A.E.B., A.A., G.S., G.S., J.S., R.T.K., N.E., E.D., G.S. and J.F. are all Agilent employees and both the Rapidfire SPE system and 6560 IMS-QTOF MS platform are Agilent instruments.

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. Two isomeric trisaccharides, melezitose and raffinose, were used to evaluate the effect of the drift gasses for mobility separation. The hybrid ion mobility Q-TOF mass analyzer used in this study consists of a low pressure uniform field drift tube apparatus coupled to a high resolution Q-TOF mass spectrometer. Conventionally, low pressure ion mobility instruments are operated using helium drift gas to obtain optimal structural information and collision cross-section (CCS) values that compare to theoretical CCS values. The instrument employed in this study uses nitrogen as the standard drift gas but also allows the utility of alternate drift gases for improved structural analysis and selectivity under certain conditions. The use of alternate drift gases with a wide range of polarizabilities allows the evaluation of mobility separation power in terms of induced dipole interactions between the drift gas and the analyte ions.

We investigated how physico-chemical properties of charged droplets are affected by the electrospray process, using simultaneous in situ measurements by laser-induced fluorescence (LIF), Fraunhofer diffraction and mass spectrometry. For this purpose, we implemented a laser-induced-fluorescence profiling setup in conjunction with a fast, high-resolution particle sizing scheme on a modified Agilent Jet Stream electrospray source coupled to a single quadrupole mass analyser. The optical setup permits us to profile the solvent fractionation and the size of the droplets as they evaporate in an electrospray plume by measuring both the angular scattering pattern and emission spectra of a solvatochromic fluorescent dye. Mass spectra are recorded simultaneously. These mass spectrometry and optical spectroscopy investigations allow us to study the relation between the observed charge-state distributions of protein anions and physico-chemical properties of evaporating droplets in the spray plume. By mixing water with methanol, a refolding of cytochrome C is observed as the water percentage increases in the plume due to the preponderant evaporation of volatile methanol.