Ion Mobility-mass Spectrometry Instruments Research Articles

Recent advances in gas phase unfolding: Instrumentation and applications.

Broader adoption of native mass spectrometry (MS) and ion mobility-mass spectrometry (IM-MS) has propelled the development of several techniques which take advantage of the selectivity, sensitivity, and speed of MS to make measurements of complex biological molecules in the gas phase. One such method, collision induced unfolding (CIU), has risen to prominence in recent years, due to its well documented capability to detect shifts in structural stability of biological molecules in response to external stimuli (e.g., mutations, stress, non-covalent interactions, sample conditions etc.). This increase in reported CIU measurements is enabled partly due to advances in IM-MS instrumentation by vendors, and alsoinnovative method development by researchers. This perspective highlights a few of these advances and concludes with a look forward toward the future of the gas phase unfolding field.

Food Fingerprinting: LC-ESI-IM-QTOF-Based Identification of Blumeatin as a New Marker Metabolite for the Detection of Origanum majorana Admixtures to O. onites/vulgare.

Oregano (Origanum vulgare and O. onites) is one of the most frequently counterfeited herbs in the world and is diluted with the leaves of a wide variety of plants. In addition to olive leaves, marjoram (O. majorana) is often used for this purpose in order to achieve a higher profit. However, apart from arbutin, no marker metabolites are known to reliably detect marjoram admixtures in oregano batches at low concentrations. In addition, arbutin is relatively widespread in the plant kingdom, which is why it is of great relevance to look for further marker metabolites in order to secure the analysis accordingly. Therefore, the aim of the present study was to use a metabolomics-based approach to identify additional marker metabolites with the aid of an ion mobility mass spectrometry instrument. The focus of the analysis was on the detection of non-polar metabolites, as this study was preceded by nuclear magnetic resonance spectroscopic investigations of the same samples based mainly on the detection of polar analytes. Using the MS-based approach, numerous marjoram specific features could be detected in admixtures of marjoram >10% in oregano. However, only one feature was detectable in admixtures of >5% marjoram. This feature was identified as blumeatin, which belongs to the class of flavonoid compounds. Initially, blumeatin was identified based on MS/MS spectra and collision cross section values using a database search. In addition, the identification of blumeatin was confirmed by a reference standard. Moreover, dried leaves of olive, myrtle, thyme, sage and peppermint, which are also known to be used to adulterate oregano, were measured. Blumeatin could not be detected in these plants, so this substance can be considered as an excellent marker compound for the detection of marjoram admixtures.

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.

Determination of Gas-Phase Ion Mobility Coefficients Using Voltage Sweep Multiplexing.

In a standard single averaged, drift tube ion mobility spectrometry (IMS) experiment, typically less than 1% of the ions are utilized, with the rest of the ions neutralizing on a closed ion gate or ion optic element. Though some efforts at lower pressures (e.g., 4Torr) have been made to address this issue by concentrating ions prior to release into a drift cell, the ion current reaching the detector during an IMS experiment is often diminished due to this lower duty cycle. Additionally, when considering the temporal nature of the drift tube IMS experiment and the trajectory of IMS towards higher resolution separations and lower duty cycles, increased detector sampling rates are another factor also which further necessitates new modes of conducting the IMS experiment. Placing this trend in context with ion mobility-mass spectrometry instruments (IM-MS), there are numerous types of mass spectrometers that are simply incompatible with the single averaged ion mobility spectrometry experiments due to timing incompatibilities (i.e., ion traps are an order of magnitude slower than the IMS experiment). However, by utilizing a dual gate ion mobility spectrometer for ion multiplexing, ion utilization efficiency can be significantly increased while creating a measurement signal that can be recorded at low sampling rates. In this work, we present the fundamental theory and first results from proof-of-concept measurements using a new type of ion multiplexing that relies on changing the electric field within the drift cell during the course of an experiment while simultaneously opening the ion gates at a constant frequency. For brevity, this mode is termed voltage sweep multiplexing (VSM). Key variables for this type of experiment are discussed and verified with measurements from traditional signal averaged experiments. Graphical Abstract .

CIUSuite: A Quantitative Analysis Package for Collision Induced Unfolding Measurements of Gas-Phase Protein Ions.

Ion mobility-mass spectrometry (IM-MS) is a technology of growing importance for structural biology, providing complementary 3D structure information for biomolecules within samples that are difficult to analyze using conventional analytical tools through the near-simultaneous acquisition of ion collision cross sections (CCSs) and masses. Despite recent advances in IM-MS instrumentation, the resolution of closely related protein conformations remains challenging. Collision induced unfolding (CIU) has been demonstrated as a useful tool for resolving isocrossectional protein ions, as they often follow distinct unfolding pathways when subjected to collisional heating in the gas phase. CIU has been used for a variety of applications, from differentiating binding modes of activation state-selective kinase inhibitors to characterizing the domain structure of multidomain proteins. With the growing utilization of CIU as a tool for structural biology, significant challenges have emerged in data analysis and interpretation, specifically the normalization and comparison of CIU data sets. Here, we present CIUSuite, a suite of software modules designed for the rapid processing, analysis, comparison, and classification of CIU data. We demonstrate these tools as part of a series of workflows for applications in comparative structural biology, biotherapeutic analysis, and high throughput screening of kinase inhibitors. These examples illustrate both the potential for CIU in general protein analysis as well as a demonstration of best practices in the interpretation of CIU data.

Utilising ion mobility-mass spectrometry to interrogate macromolecules: Factor H complement control protein modules 10–15 and 19–20 and the DNA-binding core domain of tumour suppressor p53

Ion mobility-mass spectrometry (IM-MS) has been used to study the gas-phase structures of three proteins: the DNA-binding core domain of p53 (amino acid residues 94–312) and two fragments of complement control protein factor H (regions 10–15 and 19–20) which are involved in important cellular processes. We report collision cross-sections as a function of charge state for these systems, following electrospray ionisation from ‘native’ conditions. The DNA-binding core domain of p53 contains a zinc ion that is postulated to have an important role in retaining functionality. We have chelated the zinc from the protein using phenanthroline and observed the conformational change in collision cross-section. Factor H region 10–15 has been interrogated and cross-sections have been elucidated for this recombinant protein that contains a number of covalently bound N-acetylglucosamine sugar molecules. Factor H region 19–20 is proposed to be an important polyanion-binding site for the intact factor H molecule (regions 1–20, 1213 amino acid residues in total) and characterising its structure by IM-MS has provided further information to the overall structure of the Factor H protein. This work employs two different IM-MS instruments: the commercially available Waters Synapt HDMS instrument, and our Mobility Q-TOF (“MoQTOF”) developed in house. Collision cross-sections obtained from identical molecular species differ to some extent between the two instruments. We observe some good agreement and some variation, with a general trend being that systems examined on the MoQTOF possess and present more compact collision cross-sections than observed with the same species on the Synapt. We attribute these differences principally to slight variations in solution conditions, effecting solution conformations and also to the differences in the source conditions and the ion transmission to the mobility devices affecting gas-phase conformations.