Impact of breath sample collection method and length of storage of breath samples in Tedlar bags on the level of selected volatiles assessed using gas chromatography-ion mobility spectrometry (GC-IMS).

The analysis of volatile organic compounds (VOCs) in exhaled air has attracted the interest of the scientific community because it provides the possibility of monitoring physiological and metabolic processes and non-invasive diagnostics of various diseases. However, this method remains underused in clinical practice as well as in research because of the lack of standardized procedures for the collection, storage and transport of breath samples, which would guarantee good reproducibility and comparability of results. The method of sampling, as well as the storage time of the breath samples in the polymer bags used for sample storage and transport, affect the composition and concentration of VOCs present in the breath samples. The aim of our study was to compare breath samples obtained using two methods with fully disposable equipment: a Haldane sampling tube intended for direct breath collection and breath samples exhaled into a transparent Tedlar bag. The second task was to monitor the stability of selected compounds of real breath samples stored in a Tedlar bag for 6 h. Gas chromatography coupled with ion mobility spectrometry (GC-IMS) implemented in the BreathSpec®device was used to analyse exhaled breath. Our results showed a significant difference in the signal intensity of some volatiles when taking a breath sample with a Haldane tube and a Tedlar bag. Due to its endogenous origin, acetone levels were significantly higher when the Haldane tube sampler was used while elevated levels of 2-propanol and unidentified VOC (designated as VOC 3) in the Tedlar bag samples likely originated from contamination of the Tedlar bags. The VOC stability study revealed compound-specific signal intensity changes of the selected VOCs with storage time in the Tedlar bags, with some volatiles showing increasing signal intensity during storage in Tedlar bags. This limits the use of Tedlar bags only for very limited time and carefully selected purpose. Our results highlight the importance of careful design and implementation of experiments and clinical protocols to obtain relevant and reliable results.

Ultra-high-performance liquid chromatography high-resolution mass spectrometry variants for metabolomics research.

Ultra-high-performance liquid chromatography high-resolution mass spectrometry (UHPLC-HRMS) variants currently represent the best tools to tackle the challenges of complexity and lack of comprehensive coverage of the metabolome. UHPLC offers flexible and efficient separation coupled with high-sensitivity detection via HRMS, allowing for the detection and identification of a broad range of metabolites. Here we discuss current common strategies for UHPLC-HRMS-based metabolomics, with a focus on expanding metabolome coverage.

Remodeling nanoDESI Platform with Ion Mobility Spectrometry to Expand Protein Coverage in Cancerous Tissue.

Nanospray desorption electrospray ionization mass spectrometry is an ambient ionization technique that is capable of mapping proteins in tissue sections. However, high-abundant molecules or isobaric interference in biological samples hampers its broad applications in probing low-abundant proteins. To address this challenge, herein we demonstrated an integrated module that coupled pneumatic-assisted nanospray desorption electrospray ionization mass spectrometry with high-field asymmetric ion mobility spectrometry. Using this module to analyze mouse brain sections, the protein coverage was significantly increased. This improvement allowed the mapping of low-abundant proteins in tissue sections with a 5 µm spatial resolution enabled by computationally assisted fusion with optical microscopic images. Moreover, the module was successfully applied to characterize melanoma in skin tissues based on the enhanced protein profiles. The results suggested that this integrating module will be potentially applied to discover novel proteins in cancers.

Developments in tandem ion mobility mass spectrometry.

Ion Mobility (IM) coupled to mass spectrometry (MS) is a useful tool for separating species of interest out of small quantities of heterogenous mixtures via a combination of m/z and molecular shape. While tandem MS instruments are common, instruments which employ tandem IM are less so with the first commercial IM-MS instrument capable of multiple IM selection rounds being released in 2019. Here we explore the history of tandem IM instruments, recent developments, the applications to biological systems and expected future directions.

Assessing Collision Cross Section Calibration Strategies for Traveling Wave-Based Ion Mobility Separations in Structures for Lossless Ion Manipulations.

The collision cross section (CCS) is an important property that aids in the structural characterization of molecules. Here, we investigated the CCS calibration accuracy with traveling wave ion mobility spectrometry (TWIMS) separations in structures for lossless ion manipulations (SLIM) using three sets of calibrants. A series of singly negatively charged phospholipids and bile acids were calibrated in nitrogen buffer gas using two different TW waveform profiles (square and sine) and amplitudes (20, 25, and 30 V0-p). The calibration errors for the three calibrant sets (Agilent tuning mixture, polyalanine, and one assembled in-house) showed negligible differences using a sine-shaped TW waveform. Calibration errors were all within 1-2% of the drift tube ion mobility spectrometry (DTIMS) measurements, with lower errors for sine waveforms, presumably due to the lower average and maximum fields experienced by ions. Finally, ultrahigh-resolution multipass (long path length) SLIM TWIMS separations demonstrated improved CCS calibration for phospholipid and bile acid isomers.

Distinguishing between long-term-stored and fresh chili pepper powder through fingerprinting of volatiles by headspace capillary-gas chromatography-ion mobility spectrometry.

Long-term storage of chili pepper powder results in physicochemical and microbiological changes that decrease its commercial value; these changes occur owing to fungal growth and production of off-flavor compounds. Herein, long-term-stored chili pepper powder (LSCPP) and fresh chili pepper powder (FCPP) were analyzed using internal transcribed spacer sequencing and volatile organic compound fingerprinting by headspace capillary-gas chromatography-ion mobility spectrometry. Fungal analysis detected only Xeromyces bisporus with high accuracy in all the analyzed LSCPP samples. However, the proliferation of X. bisporus on nonspecific spots complicated the distinguishing process between the two groups based solely on fungal analysis. Therefore, nine compounds (three ketones, one alcohol, two aldehydes, one ester, one furan, and one sulfur compound) obtained by autoxidation and fungal metabolism were selected as potential markers for distinguishing LSCPP and FCPP. These above-mentioned substances, which were confirmed as off-flavor species owing to "stale" odor, emitted lipid fragrance and were used to successfully distinguish LSCPP from FCPP using principal component analysis and linear discriminant analysis. PRACTICAL APPLICATION: According to the research results, it was possible to discriminate between long-term stored and fresh chili pepper powders using nine VOC markers for quality control in industry. In addition, the fungus generated from long-term storage of chili pepper powder was Xeromyces bisporus, which was confirmed to be safe for intake because it does not form secondary toxic metabolites.

Resolution of Isomeric Mixtures in Ion Mobility Using a Combined Demultiplexing and Peak Deconvolution Technique.

A combined data acquisition and data processing strategy for improving the sensitivity and resolution of ion mobility measurements is described. This strategy is implemented on a commercially available drift tube ion mobility-mass spectrometry (IM-MS) instrument and utilizes both an existing ion multiplexing strategy to achieve up to an 8-fold gain in ion signal and a new postacquisition data reconstruction technique, termed "high resolution demultiplexing" (HRdm), to improve resolution in the ion mobility dimension. A series of isomeric mixtures were qualitatively investigated with HRdm, including biologically relevant lipids and carbohydrates, which were successfully resolved by HRdm, including two monosaccharide regioisomers which differed in drift time by only 0.8%. For a complex trisaccharide isomer mixture, HRdm was able to resolve 5 out of 6 components. An analysis of two-peak resolution (Rpp) and peak-to-peak separation (ΔP) indicated that HRdm performs with an effective resolving power (Rp) of between 180 to 250 for the highest deconvolution settings. Overall analysis times and drift time measurement precision were found to be unaffected between standard and HRdm processed data sets, which allowed statistically identical collision cross section values to be directly determined from all ion mobility spectra.

Sensing Precursors of Illegal Drugs-Rapid Detection of Acetic Anhydride Vapors at Trace Levels Using Photoionization Detection and Ion Mobility Spectrometry.

Sensitive real-time detection of vapors produced by the precursors, reagents and solvents used in the illegal drugs manufacture represents a priority nowadays. Acetic anhydride (AA) is the key chemical used as acetylation agent in producing the illegal drugs heroin and methaqualone. This study was directed towards quick detection and quantification of AA in air, using two fast and very sensitive analytical techniques: photoionization detection (PID) and ion mobility spectrometry (IMS). Results obtained indicated that both PID and IMS can sense AA at ultra-trace levels in air, but while PID produces a non-selective response, IMS offers richer information. Ion mobility spectrometric response in the positive ion mode presented one product ion, at reduced ion mobility K0 of 1.89 cm2 V-1 s-1 (almost overlapped with positive reactant ion peak), while in the negative ion mode two well separated product ions, with K0 of 1.90 and 1.71 cm2 V-1 s-1, were noticed. Our study showed that by using a portable, commercial IMS system (model Mini IMS, I.U.T. GmbH Berlin) AA can be easily measured at concentrations of 0.05 ppmv (0.2 mg m-3) in negative ion mode. Best selectivity and sensitivity of the IMS response were therefore achieved in the negative operation mode.

Rapid determination of intraoperative blood propofol concentration in operating theatre by dopant-enhanced neutral release and negative photoionization ion mobility spectrometry.

The concentration of propofol in blood is an important indicator for anesthesiologists to monitor and regulate the anesthesia depth of patients during surgery. Herein, a negative photoionization ion mobility spectrometry with acetone as the dopant was developed for rapid and direct determination of intraoperative blood propofol concentration in the operating theatre. High concentration of acetone molecules in the carrier gas was used not only to enhance neutral desorption and release free propofol molecules from the whole blood, but also to increase the intensity of reactant O2- and reduce the amount of non-reactive CO3- ions simultaneously, which allowed to measure trace propofol in less than 2 min without any tedious pretreatment. Under optimized conditions, a linear calibration curve of propofol was obtained with the range of 0.5-20 ng µL-1 and with a limit of detection of 0.14 ng µL-1, which met the clinical requirements and correlated well with standard HPLC methods. Finally, the method was applied to detect intraoperative blood propofol concentration in nearly 100 surgical patients, demonstrating its excellent detection capability and facilitating the study of propofol pharmacokinetics.

Optimized Workflow for Multiplexed Phosphorylation Analysis of TMT-Labeled Peptides Using High-Field Asymmetric Waveform Ion Mobility Spectrometry.

Phosphorylation is a post-translational modification with a vital role in cellular signaling. Isobaric labeling-based strategies, such as tandem mass tags (TMT), can measure the relative phosphorylation states of peptides in a multiplexed format. However, the low stoichiometry of protein phosphorylation constrains the depth of phosphopeptide analysis by mass spectrometry. As such, robust and sensitive workflows are required. Here we evaluate and optimize high-Field Asymmetric waveform Ion Mobility Spectrometry (FAIMS) coupled to Orbitrap Tribrid mass spectrometers for the analysis of TMT-labeled phosphopeptides. We determined that using FAIMS-MS3 with three compensation voltages (CV) in a single method (e.g., CV = -40/-60/-80 V) maximizes phosphopeptide coverage while minimizing inter-CV overlap. Furthermore, consecutive analyses using MSA-CID (multistage activation collision-induced dissociation) and HCD (higher-energy collisional dissociation) fragmentation at the MS2 stage increases the depth of phosphorylation analysis. The methodology and results outlined herein provide a template for tailoring optimized FAIMS-based methods.

Fundamentals of Ion Mobility-Mass Spectrometry for the Analysis of Biomolecules.

Ion mobility-mass spectrometry (IM-MS) combines complementary size- and mass-selective separations into a single analytical platform. This chapter provides context for both the instrumental arrangements and key application areas that are commonly encountered in bioanalytical settings. New advances in these high-throughput strategies are described with description of complementary informatics tools to effectively utilize these data-intensive measurements. Rapid separations such as these are especially important in systems, synthetic, and chemical biology in which many small molecules are transient and correspond to various biological classes for integrated omics measurements. This chapter highlights the fundamentals of IM-MS and its applications toward biomolecular separations and discusses methods currently being used in the fields of proteomics, lipidomics, and metabolomics.

Traveling-Wave-Based Electrodynamic Switch for Concurrent Dual-Polarity Ion Manipulations in Structures for Lossless Ion Manipulations.

We describe the development of a dual-polarity traveling-wave (TW) structures for lossless ion manipulations (SLIM) ion mobility spectrometry (IMS) device capable of switching both positive and negative ions that are traveling simultaneously along the same path to different regions of the SLIM. Through simulations, the routing efficiency of the SLIM TW switch was compared to a SLIM direct-current-based (DC) switch developed previously for IMS-MS. We also report on the initial experimental evaluation of a dual-polarity SLIM platform, which uses the TW-based ion switch to achieve higher resolution multipass serpentine ultralong path with extended routing (SUPER) IMS separations. Overall, these results show that the dual-polarity TW switch is not only as effective as DC switching in terms of routing efficiency but also is agnostic to the polarity of the ions being routed.

Covalent Cross-Linking as an Enabler for Structural Mass Spectrometry.

The number of studies referring to the structural elucidation of intact biomolecular systems using mass spectrometry techniques has gradually increased in the post-2000s literature topics. As part of native mass spectrometry, this domain capitalizes on the kinetic trapping of physiological folds in view of probing solution-like conformational properties of isolated molecules or complexes after their electrospray transfer to the gas phase. Despite its efficiency for a wide array of analytes, this approach is expected to be pushed to its limits when considering highly dynamic systems or when dealing with nonideal operating conditions. To circumvent these limitations, we challenge the adequacy of an original strategy based on cross-linkers to improve the gas-phase stability of isolated proteins and ensure the preservation of folded conformations when measuring with strong transmission voltages, by spraying from denaturing solvents, or trapping for extended periods of time. Tested on cytochrome c, myoglobin, and ß-lactoglobulin cross-linked using BS3, we validated the process as structurally nonintrusive in solution using far-ultraviolet circular dichroism and unraveled the preservation of folded conformations showing better resilience to denaturation on cross-linked species using ion mobility. The resulting collision cross sections were found in agreement with the native fold, and a preservation of the proteins' secondary and tertiary structures was evidenced using molecular dynamics simulations. Our results provide new insights concerning the fate of electro-sprayed cross-linked conformers in the gas phase, while constituting promising evidence for the validation of this technique as part of future structural mass spectrometry workflows.

A needle-to-post air discharge ion source in tandem with FAIMS system.

A needle-to-post ionization source was designed for high-field asymmetric waveform ion mobility spectrometry (FAIMS). The needle-to-post ion source includes asymmetric electrode comprised of a copper post with a diameter of 2 mm and a stainless-steel needle with 200-µm tip radius and length of 28 mm. With the discharge voltage of -5.6 kV and N2 gas flow, glow discharge was realized at atmospheric pressure. The mass spectra of ionized ions about acetone, ethanol and ethyl acetate were gotten by Thermo Scientific LTQ XL ion trap mass spectrometer (MS). The MS experimental results show that the main ions are protonated and dimer ions. The needle-to-post ion source was mounted on the FAIMS system and FAIMS spectra are gotten successfully. Separation of p-xylene, o-xylene and m-xylene was realized. It shows that the needle-to-post electrode could be used as the ion source in a FAIMS system.

Miniaturized Ion Mobility Spectrometer with a Dual-Compression Tristate Ion Shutter for On-Site Rapid Screening of Fentanyl Drug Mixtures.

The hidden presence of fentanyl and new psychoactive substances in established illicit drugs has led to a great number of unintentional fatal overdoses, justifying the urgent need for portable tools for the rapid screening of various drugs. Ion mobility spectrometry, as a rapid detection technique, has been widely used in detecting illicit drugs, whereas it is insufficiently sensitive for bigger ions due to the mobility discrimination of an ion shutter. In this work, a miniaturized ion mobility spectrometer with an inner diameter of 14 mm and a drift length of 38.9 mm and equipped with a novel dual-compression tristate ion shutter has been developed, which could greatly reduce the mobility discrimination by compressing the ion packet twice during the injection process. The best gating performance of the dual-compression tristate ion shutter was about three times as high as that of the tristate ion shutter and twice as high as that of the Tyndall-Powell shutter. For example, the peak height was increased by 150 or 40% when the resolving power was the same, whereas the resolution of two neighboring peaks was improved by 46 or 19% when the peak heights were the same. In screening of fentanyl drug mixtures, the new shutter enhanced the identification accuracy of constituents by making peaks of slow dimer ions and complex ions observable. Besides, the new shutter helped to achieve high sensitivity for drug ions spreading a wide ion mobility range from 0.644 to 2.032 cm2 s-1 V-1, demonstrating its potential use in the analysis of other mixtures and the detection of large ions.

Statistical analysis of MCC-IMS data for two group comparisons-an exemplary study on two devices.

The Multi-capillary-column-Ion-mobility-spectrometry (MCC-IMS) technology for measuring breath gas can be used for distinguishing between healthy and diseased subjects or between different types of diseases. The statistical methods for classifying the corresponding breath samples typically neglects potential confounding clinical and technical variables, reducing both accuracy and generalizability of the results. Especially measuring samples on different technical devices can heavily influence the results. We conducted a controlled breath gas study including 49 healthy volunteers to evaluate the effect of the variables sex, smoking habits and technical device. Every person was measured twice, once before and once after consuming a glass of orange juice. The two measurements were obtained on two different devices. The evaluation of the MCC-IMS data regarding metabolite detection was performed once using the software VisualNow, which requires manual interaction, and once using the fully automated algorithm SGLTR-DBSCAN. We present statistical solutions, peak alignment and scaling, to adjust for the different devices. For the other potential confounders sex and smoking, in our study no significant influence was identified.

Rapid characterization of structural and functional similarity for a candidate bevacizumab (Avastin) biosimilar using a multipronged mass-spectrometry-based approach.

The ongoing shift from small molecule drugs to protein therapeutics in the pharmaceuticals industry presents a considerable challenge to generic drug developers who are increasingly required to demonstrate biosimilarity for biological macromolecules, a task that is decidedly more complex than doing the same for small molecule drugs. In this work, we demonstrate a multipronged mass-spectrometry-based workflow that allows rapid and facile molecular characterization of antibody-based protein therapeutics, applied to biosimilars development. Specifically, we use a combination of native mass spectrometry (MS), ion mobility spectrometry (IMS), and global time-resolved hydrogen deuterium exchange (HDX) to provide an unambiguous assessment of the structural, dynamic, and chemical similarity between Avastin (bevacizumab) and a biosimilar in the late stages of pre-clinical development. Minor structural and dynamic differences between the biosimilar and Avastin, and between lots of the biosimilar, were tested for functional relevance using Surface Plasmon Resonance-derived kinetic and equilibrium binding parameters.

On the Preservation of Non-covalent Peptide Assemblies in a Tandem-Trapped Ion Mobility Spectrometer-Mass Spectrometer (TIMS-TIMS-MS).

Ion mobility spectrometry-mass spectrometry (IMS-MS) has demonstrated the ability to characterize structures of weakly-bound peptide assemblies. However, these assemblies can potentially dissociate during the IMS-MS measurement if they undergo energetic ion-neutral collisions. Here, we investigate the ability of tandem-trapped ion mobility spectrometry-mass spectrometry (TIMS-TIMS-MS) to retain weakly-bound peptide assemblies. We assess ion heating and dissociaton in the tandem-TIMS instrument using bradykinin and its assemblies as reference systems. Our data indicate that non-covalent bradykinin assemblies are largely preserved in TIMS-TIMS under carefully selected operating conditions. Importantly, we observe quadruply-charged bradykinin tetramers, which attests to the "softness" of our instrument. Graphical Abstract.

A Modified Traveling Wave Ion Mobility Mass Spectrometer as a Versatile Platform for Gas-Phase Ion-Molecule Reactions.

Taken individually, chemical labeling and mass spectrometry are two well-established tools for the structural characterization of biomolecular complexes. A way to combine their respective advantages is to perform gas-phase ion-molecule reactions (IMRs) inside the mass spectrometer. This is, however, not so well developed because of the limited range of usable chemicals and the lack of commercially available IMR devices. Here, we modified a traveling wave ion mobility mass spectrometer to enable IMRs in the trapping region of the instrument. Only one minor hardware modification is needed to allow vapors of a variety of liquid reagents to be leaked into the trap traveling wave ion guide of the instrument. A diverse set of IMRs can then readily be performed without any loss in instrument performance. We demonstrate the advantages of implementing IMR capabilities in general, and to this quadrupole-ion mobility-time-of-flight (Q-IM-TOF) mass spectrometer in particular, by exploiting the full functionality of the instrument, including mass selection, ion mobility separation, and post-mobility fragmentation. The potential to carry out gas-phase IMR kinetics experiments is also illustrated. We demonstrate the versatility of the setup using gas-phase IMRs of established utility for biological mass spectrometry, including hydrogen-deuterium exchange, ion-molecule proton transfer reactions, and covalent modification of DNA anions using trimethylsilyl chloride.

Accurate Quantitative Proteomic Analyses Using Metabolic Labeling and High Field Asymmetric Waveform Ion Mobility Spectrometry (FAIMS).

Stable isotope labeling by amino acids in cell culture (SILAC) is routinely used to profile changes in protein and peptide abundance across different experimental paradigms. As with other quantitative proteomic approaches, the detection of peptide isotopomers can be limited by the presence of interference ions that ultimately affect the quality of quantitative measurements. Here, we evaluate high field asymmetric waveform ion mobility spectrometry (FAIMS) to improve the accuracy and dynamic range of quantitative proteomic analyses using SILAC. We compared quantitative measurements for tryptic digests of isotopically labeled protein extracts mixed in different ratios using LC-MS/MS with and without FAIMS. To further reduce sample complexity, we also examined the improvement in quantitative measurements when combining strong cation exchange (SCX) fractionation prior to LC-MS/MS analyses. Using the same amount of sample consumed, analyses performed using FAIMS provided more than 30% and 200% increase in the number of quantifiable peptides compared to LC-MS/MS performed with and without SCX fractionation, respectively. Furthermore, FAIMS reduced the occurrence of interfering isobaric ions and improved the accuracy of quantitative measurements. We leveraged the application of FAIMS in phosphoproteomic analyses to profile dynamic changes in protein phosphorylation in HEK293 cells subjected to heat shock for periods up to 20 min. In addition to the enhanced phosphoproteomic coverage, FAIMS also provided the ability to separate phosphopeptide isomers that often coelute and can be misassigned in conventional LC-MS/MS experiments.