S.O. is thankful to the French National Research Agency for funding his Ph.D. (grant ANR-18-CE29-0006).

The authors have no conflict of interest to disclose.

The SELECT SERIES Cyclic IMS is a powerful tool that allows selecting a defined ion population&#8212;of a given m/z and ion mobility&#8212;without the need for upstream chromatographic separation. The instrument affords the possibility of generating a bidimensional fragmentation map of this ion population, from which both MS/MS and IMS/IMS spectra can be extracted. However, the user must note several critical points that require attention during the experimental process.
First, the us...

The complete chemical characterization of biomolecules is key to understanding their underlying biological and functional properties. To this end, &#34;omics&#34; sciences have developed in recent years, aiming for the large-scale characterization of chemical structures at biological concentrations. In proteomics and metabolomics, MS has become a core tool to unravel the structural heterogeneity found in biological media-notably thanks to its sensitivity and ability to provide structural information through tandem MS (MS/MS). In MS/MS strategies, an ion is selected according to its mass, then fragmented, and finally, the masses of its fragments are acquired to establish a fingerprint of the molecule. MS/MS spectra can, in particular, be used to match spectral databases1,2, or tentatively reconstruct the parent structures3,4. Under the assumption that similar spectra belong to similar compounds, MS/MS data can also be used to build molecular networks (MNs) connecting related species through a similarity score5,6.
However, because of the inherent property of MS to detect the mass-to-charge ratio (m/z) of ions, the technique is blind to a number of structural features that fall within the range of (stereo)isomerism. For example, carbohydrates are made of several monosaccharide subunits, many of which are stereoisomers or even epimers (e.g., Glc vs. Gal or Glc vs. Man). These subunits are linked by glycosidic bonds, which can differ by the position of the linkage (regioisomerism) and the steric configuration of the anomeric carbon (anomerism). These characteristics make it difficult for standalone MS to distinguish between carbohydrate isomers7, and only regioisomerism can be addressed using high-energy activation methods8,9,10. Although derivatization is an option to disrupt the equivalence of stereoisomeric groups11, it requires extensive sample preparation. Another, more straightforward option is to couple MS with an analytical dimension sensitive to isomerism, such as IMS.
Because this protocol is designed for users who are already familiar with the basic concepts of IMS, and because detailed reviews are available elsewhere12,13, only a brief overview of the principles of IMS is given here. IMS is a gas-phase separation method that relies on the interaction of ions with a buffer gas and an electric field, ultimately separating ions according to their gas-phase conformations. Different principles of IMS coupled to MS can be found on commercial instruments: some operate at alternating high and low electric fields (field asymmetric IMS, FAIMS), while most operate within the low field limit&#8212;notably drift tube IMS (DTIMS, linearly decreasing electric field), traveling wave IMS (TWIMS, symmetric potential waves), and trapped IMS (TIMS, high flow of buffer gas trapping ions against electric fields)13. The low-field methods allow access to a so-called CCS, a property of the ion-gas pair that represents the surface (in &#197;2 or nm2) of the ion that interacts with the buffer gas during the separation. CCS is theoretically instrument-independent and is thus useful to generate data that can be reproduced between different laboratories14. Ion mobility separations can be impacted by various parameters and, notably, by fluctuations of the gas pressure and gas temperature in the mobility cell. The CCS calibration is a way to remedy this, as both the calibrant and the species of interest will be similarly affected13. However, it is mandatory to install the instrument in a temperature-controlled room and to have a reliable gas pressure control system.
An interesting evolution of IMS is IMS/IMS, which was first introduced in 2006 by Clemmer&#39;s group as an analog of MS/MS15,16. In IMS/IMS, an ion of interest is selectively isolated based on its ion mobility; it is then activated (until possible fragmentation), and a new IMS analysis of the activated ion or fragments is performed. In the first instrumental design, two IMS cells were put in series, separated by an ion funnel where the activation stood. Since then, although a number of IMS/IMS setups were proposed (for a review, see Eldrid and Thalassinos17), the first commercial mass spectrometer with IMS/IMS capability only became available in 201918. This instrument substantially improved the initial concept by combining it with another technological breakthrough: a cyclic design of the IMS cell.
The cyclic IMS cell theoretically allows increasing near-infinitely the drift path length and, thus, the resolving power of the instrument19. This was achieved by means of a particular instrument geometry, where the cyclic TWIMS cell is placed orthogonally to the main ion optical axis. A multifunction array region at the entrance of the IMS cell allows controlling the direction of the ion path: (i) sending ions sideways for IMS separation, (ii) forward for MS detection, or (iii) backward from the IMS cell to be stored in a prearray cell. From this prearray store cell, the ions can be activated and the fragments reinjected in the IMS cell for ion mobility measurement, an approach that has been successfully used to characterize stereoisomers20. Ultimately, the collected data contain ion mobility and m/z information for the precursor and its fragments.
In a recent publication that used this cyclic design for glycan analyses (Ollivier et al.21), we showed that the mobility profile of the fragments contained in such IMS/IMS data acts as a fingerprint of a biomolecule that can be used in a molecular networking strategy. The resulting network, called IM-MN, led to the organization of glycomics datasets in a structurally relevant way, whereas the network built solely from MS/MS data (MS-MN) revealed little information. To complement this publication and help Cyclic IMS users implement this workflow, this protocol provides a complete description of the protocol used to collect the data. This protocol focuses only on the generation of the IMS/IMS data that users can then use to build IM-MN networks (see21)&#8212;or for any other application of their choice. Building of IM-MN will not be considered herein, as protocols for molecular networking are already available22. The crucial points that must be followed to generate valuable and reproducible IMS/IMS acquisitions are highlighted. Taking the example of one of the oligosaccharides studied by Ollivier et al.21, the following steps are detailed: (i) sample preparation, (ii) tuning of the Cyclic IMS instrument, (iii) automated peak-picking of the data, and (iv) CCS calibration.

<table><tbody><tr><td>33-&amp;alpha;-L- plus 23-&amp;alpha;-L-Arabinofuranosyl-xylotetraose (XA3XX/XA2XX) mixture</td><td>Megazyme Ltd., Wicklow, Ireland</td><td>O-XAXXMIX</td><td>XA&lt;sup&gt;2&lt;/sup&gt;XX + XA&lt;sup&gt;3&lt;/sup&gt;XX mixture</td></tr><tr><td>33-&amp;alpha;-L-Arabinofuranosyl-xylotetraose (XA3XX)</td><td>Megazyme Ltd., Wicklow, Ireland</td><td>O-XA3XX</td><td>Pure XA&lt;sup&gt;3&lt;/sup&gt;XX standard</td></tr><tr><td>Eppendorf Safe-Lock Tubes, 1.5 mL, Eppendorf Quality, colorless, 1,000 tubes</td><td>Eppendorf, Hamburg, Germany</td><td>0030120086</td><td>Used to prepare the carbohydrate stock solution and dilution</td></tr><tr><td>FALCON 50 mL Polypropylene Conical Tube 30 x 115 mm</td><td>Corning Science M&amp;eacute;xico S.A. de C.V., Reynosa, Tamaulipas, Mexico</td><td>352070</td><td>Used to prepare the aqueous stock solution of 100 mM LiCl</td></tr><tr><td>Lithium Chloride (ACS reagent, &amp;ge;99 %)</td><td>Sigma-Aldrich Inc., Saint Quentin Fallavier, France</td><td>310468</td><td>Used to dope the sample with lithium</td></tr><tr><td>Major Mix IMS/Tof Calibration Kit</td><td>Waters Corp., Wilmslow, UK</td><td>186008113</td><td>Calibration solution for MS and IMS</td></tr><tr><td>MassLynx 4.2 SCN1016 Release 6 (Waters Embedded Analyser Platform for Cyclic IMS 2.9.1 Release 9)</td><td>Waters Corp., Wilmslow, UK</td><td>721022377</td><td>Cyclic IMS vendor software for instrument control and data processing</td></tr><tr><td>Methanol for HPLC PLUS Gradient grade</td><td>Carlo-Erba Reagents, Val de Reuil, France</td><td>412383</td><td>High-purity solvent</td></tr><tr><td>MS Leucine Enkephaline Kit</td><td>Waters Corp., Wilmslow, UK</td><td>700002456</td><td>Reference compound used for tuning of the mass spectrometer</td></tr><tr><td>SCHOTT DURAN 100 mL borosilicate glass bottle</td><td>VWR INTERNATIONAL, Radnor, Pennsylvania, US</td><td>218012458</td><td>Used to prepare the solution of 500 &amp;micro;M LiCl in 50:50 MeOH/Water</td></tr><tr><td>SELECT SERIES Cyclic IMS</td><td>Waters Corp., Wilmslow, UK</td><td>186009432</td><td>Ion mobility-mass spectrometer equipped with a cylic IMS cell</td></tr><tr><td>Website: http://mzmine.github.io/</td><td>MZmine Development Team</td><td>-</td><td>Link to download the MZmine software</td></tr><tr><td>Website: https://github.com/siollivier/IM-MN</td><td>INRAE, UR BIA, BIBS Facility, Nantes, France</td><td>-</td><td>Link to an in-house R script containing a CCS calibration function</td></tr></tbody></table>

using a cyclic ion mobility spectrometer for tandem ion mobility experiments

Accurate characterization of chemical structures is important to understand their underlying biological mechanisms and functional properties. Mass spectrometry (MS) is a popular tool but is not always sufficient to completely unveil all structural features. For example, although carbohydrates are biologically relevant, their characterization is complicated by numerous levels of isomerism. Ion mobility spectrometry (IMS) is an interesting complement because it is sensitive to ion conformations and, thus, to isomerism. 
Furthermore, recent advances have significantly improved the technique: the last generation of Cyclic IMS instruments offers additional capabilities compared to linear IMS instruments, such as an increased resolving power or the possibility to perform tandem ion mobility (IMS/IMS) experiments. During IMS/IMS, an ion is selected based on its ion mobility, fragmented, and reanalyzed to obtain ion mobility information about its fragments. Recent work showed that the mobility profiles of the fragments contained in such IMS/IMS data can act as a fingerprint of a particular glycan and can be used in a molecular networking strategy to organize glycomics datasets in a structurally relevant way. 
The goal of this protocol is thus to describe how to generate IMS/IMS data, from sample preparation to the final Collision Cross Section (CCS) calibration of the ion mobility dimension that yields reproducible spectra. Taking the example of one representative glycan, this protocol will show how to build an IMS/IMS control sequence on a Cyclic IMS instrument, how to account for this control sequence to translate IMS arrival time into drift time (i.e., the effective separation time applied to the ions), and how to extract the relevant mobility information from the raw data. This protocol is designed to clearly explain the critical points of an IMS/IMS experiment and thus help new Cyclic IMS users perform straightforward and reproducible acquisitions.

An arabinoxylan pentasaccharide, XA2XX, was chosen as an example to illustrate this protocol. This compound is commercially available, but only as a mixture with another arabinoxylan pentasaccharide, XA3XX (pure XA3XX is also commercially available). The structures of XA2XX and XA3XX are given in Supplemental&#160;Figure S1. As the ratio of XA2XX and XA3XX in the commercial mixture is ~50:50, a solution at 20 &#181;g/mL of the m...

Watch this Scientific Journal Video about Using a Cyclic Ion Mobility Spectrometer for Tandem Ion Mobility Experiments at JoVE.com

Using a Cyclic Ion Mobility Spectrometer for Tandem Ion Mobility Experiments

Ion mobility spectrometry (IMS) is an interesting complement to mass spectrometry for the characterization of biomolecules, notably because it is sensitive to isomerism. This protocol describes a tandem IMS (IMS/IMS) experiment, which allows the isolation of a molecule and the generation of the mobility profiles of its fragments.

Accurate characterization of chemical structures is important to understand their underlying biological mechanisms and functional properties. Mass ...

using-cyclic-ion-mobility-spectrometer-for-tandem-ion-mobility

Research

JoVE Journal

Chemistry

4.2K Views.  INRAE, UR BIA, F-44316 Nantes, France. Ion mobility spectrometry (IMS) is an interesting complement to mass spectrometry for the characterization of biomolecules, notably because it is sensitive to isomerism. This protocol describes a tandem IMS (IMS/IMS) experiment, which allows the isolation of a molecule and the generation of the mobility profiles of its fragments. 

Video: Using a Cyclic Ion Mobility Spectrometer for Tandem Ion Mobility Experiments

Ion Exchange Chromatography (IEX) Coupled to Multi-angle Light Scattering (MALS) for Protein Separation and Characterization

Ion-exchange chromatography with multi-angle light scattering (IEX-MALS) is a powerful method for protein separation and characterization. The combination of the high-specificity separation technique IEX with the accurate molar mass analysis achieved by MALS allows the characterization of heterogeneous protein samples, including mixtures of oligomeric forms or protein populations, even with very similar molar masses. Therefore, IEX-MALS provides an additional level of protein characterization and is complementary to the standard size-exclusion chromatography with multi-angle light scattering (SEC-MALS) technique.
Here we describe a protocol for a basic IEX-MALS experiment and demonstrate this method on bovine serum albumin (BSA). IEX separates BSA to its oligomeric forms allowing a molar mass analysis by MALS of each individual form. Optimization of an IEX-MALS experiment is also presented and demonstrated on BSA, achieving excellent separation between BSA monomers and larger oligomers. IEX-MALS is a valuable technique for protein quality assessment since it provides both fine separation and molar mass determination of multiple protein species that exist in a sample.

Ion Exchange Chromatography IEX Coupled to Multi-angle Light Scattering MALS for Protein Separation and Characterization

This protocol describes the use of high-specificity ion-exchange chromatography with multi-angle light scattering for an accurate molar mass determination of proteins, protein complexes, and peptides in a heterogeneous sample. This method is valuable for quality assessment, as well as for the characterization of native oligomers, charge variants, and mixed-protein samples.

Ion-exchange chromatography with multi-angle light scattering (IEX-MALS) is a powerful method for protein separation and characterization. The combination ...

Biochemistry

Analyzing Protein Architectures and Protein-Ligand Complexes by Integrative Structural Mass Spectrometry

Proteins are an important class of biological macromolecules that play many key roles in cellular functions including gene expression, catalyzing metabolic reactions, DNA repair and replication. Therefore, a detailed understanding of these processes provides critical information on how cells function. Integrative structural MS methods offer structural and dynamical information on protein complex assembly, complex connectivity, subunit stoichiometry, protein oligomerization and ligand binding. Recent advances in integrative structural MS have allowed for the characterization of challenging biological systems including large DNA binding proteins and membrane proteins. This protocol describes how to integrate diverse MS data such as native MS and ion mobility-mass spectrometry (IM-MS) with molecular dynamics simulations to gain insights into a helicase-nuclease DNA repair protein complex. The resulting approach provides a framework for detailed studies of ligand binding to other protein complexes involved in important biological processes.

Mass spectrometry (MS) has emerged as an important tool for the investigation of structure and dynamics of macromolecular assemblies. Here, we integrate MS-based approaches to interrogate protein complex formation and ligand binding.

Proteins are an important class of biological macromolecules that play many key roles in cellular functions including gene expression, catalyzing ...

Ion Mobility-Mass Spectrometry Techniques for Determining the Structure and Mechanisms of Metal Ion Recognition and Redox Activity of Metal Binding Oligopeptides

Electrospray ionization (ESI) can transfer an aqueous-phase peptide or peptide complex to the gas-phase while conserving its mass, overall charge, metal-binding interactions, and conformational shape. Coupling ESI with ion mobility-mass spectrometry (IM-MS) provides an instrumental technique that allows for simultaneous measurement of a peptide&#8217;s mass-to-charge (m/z) and collision cross section (CCS) that relate to its stoichiometry, protonation state, and conformational shape. The overall charge of a peptide complex is controlled by the protonation of 1) the peptide&#8217;s acidic and basic sites and 2) the oxidation state of the metal ion(s). Therefore, the overall charge state of a complex is a function of the pH of the solution that affects the peptides metal ion binding affinity. For ESI-IM-MS analyses, peptide and metal ions solutions are prepared from aqueous-only solutions, with the pH adjusted with dilute aqueous acetic acid or ammonium hydroxide. This allows for pH dependence and metal ion selectivity to be determined for a specific peptide. Furthermore, the m/z and CCS of a peptide complex can be used with B3LYP/LanL2DZ molecular modeling to discern binding sites of the metal ion coordination and tertiary structure of the complex. The results show how ESI-IM-MS can characterize the selective chelating performance of a set of alternative methanobactin peptides and compare them to the copper-binding peptide methanobactin.

Ion mobility-mass spectrometry and molecular modeling techniques can characterize the selective metal chelating performance of designed metal-binding peptides and the copper-binding peptide methanobactin. Developing new classes of metal chelating peptides will help lead to therapeutics for diseases associated with metal ion misbalance.

Electrospray ionization (ESI) can transfer an aqueous-phase peptide or peptide complex to the gas-phase while conserving its mass, overall charge, ...

Cellular Membrane Affinity Chromatography Columns to Identify Specialized Plant Metabolites Interacting with Immobilized Tropomyosin Kinase Receptor B

Chemicals synthesized by plants, fungi, bacteria, and marine invertebrates have been a rich source of new drug hits and leads. Medicines such as statins, penicillin, paclitaxel, rapamycin, or artemisinin, commonly used in medical practice, have been first identified and isolated from natural products. However, the identification and isolation of biologically active specialized metabolites from natural sources is a challenging and time-consuming process. Traditionally, individual metabolites are isolated and purified from complex mixtures, following the extraction of biomass. Subsequently, the isolated molecules are tested in functional assays to verify their biological activity. Here we present the use of cellular membrane affinity chromatography (CMAC) columns to identify biologically active compounds directly from complex mixtures. CMAC columns allow for the identification of compounds interacting with immobilized functional transmembrane proteins (TMPs) embedded in their native phospholipid bilayer environment. This is a targeted approach, which requires knowing the TMP whose activity one intends to modulate with the newly identified small molecule drug candidate. In this protocol, we present an approach to prepare CMAC columns with immobilized tropomyosin kinase receptor B (TrkB), which has emerged as a viable target for drug discovery for numerous nervous system disorders. In this article, we provide a detailed protocol to assemble the CMAC column with immobilized TrkB receptors using neuroblastoma cell lines overexpressing TrkB receptors. We further present the approach to investigate the functionality of the column and its use in the identification of specialized plant metabolites interacting with TrkB receptors.

The protocol describes the preparation of cell membrane affinity chromatography (CMAC) columns with immobilized cell membrane fragments containing functional transmembrane tropomyosin kinase receptor B proteins. The use of CMAC columns in the identification of specialized plant metabolites interacting with these receptors and present in complex natural mixtures is also explained.

Chemicals synthesized by plants, fungi, bacteria, and marine invertebrates have been a rich source of new drug hits and leads. Medicines such as statins, ...

Thermochemical Studies of Ni(II) and Zn(II) Ternary Complexes Using Ion Mobility-Mass Spectrometry

This article describes an experimental protocol using electrospray-ion mobility-mass spectrometry (ES-IM-MS) and energy-resolved threshold collision-induced dissociation (TCID) to measure the thermochemistry of the dissociation of negatively-charged [amb+M(II)+NTA]- ternary complexes into two product channels: [amb+M(II)] + NTA or [NTA+M(II)]-&#160;+ amb, where M = Zn or Ni and NTA is nitrilotriacetic acid. The complexes contain one of the alternative metal binding (amb) heptapeptides with the primary structures acetyl-His1-Cys2-Gly3-Pro4-Tyr5-His6-Cys7 or acetyl-Asp1-Cys2-Gly3-Pro4-Tyr5-His6-Cys7, where the amino acids&#39; Aa1,2,6,7 positions are the potential metal-binding sites. Geometry-optimized stationary states of the ternary complexes and their products were selected from quantum chemistry calculations (presently the PM6 semi-empirical Hamiltonian) by comparing their electronic energies and their collision cross-sections (CCS) to those measured by ES-IM-MS. From the PM6 frequency calculations, the molecular parameters of the ternary complex and its products model the energy-dependent intensities of the two product channels using a competitive TCID method to determine the threshold energies of the reactions that relate to the 0 K enthalpies of dissociation (&#916;H0). Statistical mechanics thermal and entropy corrections using the PM6 rotational and vibrational frequencies provide the 298 K enthalpies of dissociation (&#916;H298). These methods describe an EI-IM-MS routine that can determine thermochemistry and equilibrium constants for a range of ternary metal ion complexes.

Thermochemical Studies of NiII and ZnII Ternary Complexes Using Ion Mobility-Mass Spectrometry

This article describes an experimental protocol using electrospray-ion mobility-mass spectrometry, semi-empirical quantum calculations, and energy-resolved threshold collision-induced dissociation to measure the relative thermochemistry of the dissociation of related ternary metal complexes.

This article describes an experimental protocol using electrospray-ion mobility-mass spectrometry (ES-IM-MS) and energy-resolved threshold ...

T-wave Ion Mobility-mass Spectrometry: Basic Experimental Procedures for Protein Complex Analysis

Ion mobility (IM) is a method that measures the time taken for an ion to travel through a pressurized cell under the influence of a weak electric field. The speed by which the ions traverse the drift region depends on their size: large ions will experience a greater number of collisions with the background inert gas (usually N2) and thus travel more slowly through the IM device than those ions that comprise a smaller cross-section. In general, the time it takes for the ions to migrate though the dense gas phase separates them, according to their collision cross-section (&Omega;). 
Recently, IM spectrometry was coupled with mass spectrometry and a traveling-wave (T-wave) Synapt ion mobility mass spectrometer (IM-MS) was released. Integrating mass spectrometry with ion mobility enables an extra dimension of sample separation and definition, yielding a three-dimensional spectrum (mass to charge, intensity, and drift time). This separation technique allows the spectral overlap to decrease, and enables resolution of heterogeneous complexes with very similar mass, or mass-to-charge ratios, but different drift times. Moreover, the drift time measurements provide an important layer of structural information, as &Omega; is related to the overall shape and topology of the ion. The correlation between the measured drift time values and &Omega; is calculated using a calibration curve generated from calibrant proteins with defined cross-sections1.
The power of the IM-MS approach lies in its ability to define the subunit packing and overall shape of protein assemblies at micromolar concentrations, and near-physiological conditions1. Several recent IM studies of both individual proteins2,3 and non-covalent protein complexes4-9, successfully demonstrated that protein quaternary structure is maintained in the gas phase, and highlighted the potential of this approach in the study of protein assemblies of unknown geometry. Here, we provide a detailed description of IMS-MS analysis of protein complexes using the Synapt (Quadrupole-Ion Mobility-Time-of-Flight) HDMS instrument (Waters Ltd; the only commercial IM-MS instrument currently available)10. We describe the basic optimization steps, the calibration of collision cross-sections, and methods for data processing and interpretation. The final step of the protocol discusses methods for calculating theoretical &Omega; values. Overall, the protocol does not attempt to cover every aspect of IM-MS characterization of protein assemblies; rather, its goal is to introduce the practical aspects of the method to new researchers in the field.

Ion mobility-mass spectrometry is an emerging gas-phase technology that separates ions, based on their collision cross-section and mass. The method provides three-dimensional information on the overall topology and shape of protein complexes. Here, we outline a basic procedure for instrument setting and optimization, calibration of drift times, and data interpretation.

Ion mobility (IM) is a method that measures the time taken for an ion to travel through a pressurized cell under the influence of a weak electric field. ...

Biology

Histone Proteomic Analysis and Post-Translational Modification Characterization

Histone Modification Screening using Liquid Chromatography, Trapped Ion Mobility Spectrometry, and Time-Of-Flight Mass Spectrometry

Histone proteins are highly abundant and conserved among eukaryotes and play a large role in gene regulation as a result of structures known as posttranslational modifications (PTMs). Identifying the position and nature of each PTM or pattern of PTMs in reference to external or genetic factors allows this information to be statistically correlated with biological responses such as DNA transcription, replication, or repair. In the present work, a high-throughput analytical protocol for the detection of histone PTMs from biological samples is described. The use of complementary liquid chromatography, trapped ion mobility spectrometry, and time-of-flight mass spectrometry (LC-TIMS-ToF MS/MS) enables the separation and PTM assignment of the most biologically relevant modifications in a single analysis. The described approach takes advantage of recent developments in dependent data acquisition (DDA) using parallel accumulation in the mobility trap, followed by sequential fragmentation and collision-induced dissociation. Histone PTMs are confidently assigned based on their retention time, mobility, and fragmentation pattern.

Author Spotlight: Enhanced Histone PTM Isomer Identification Through LC-TIMS-ToF MS/MS and PASEF

An analytical workflow based on liquid chromatography, trapped ion mobility spectrometry, and time-of-flight mass spectrometry (LC-TIMS-ToF MS/MS) for high confidence and highly reproducible "bottom-up" analysis of histone modifications and identification based on principal parameters (retention time [RT], collision cross section [CCS], and accurate mass-to-charge [m/z] ratio).

Histone proteins are highly abundant and conserved among eukaryotes and play a large role in gene regulation as a result of structures known as ...

Enhanced Isomeric Separation of Ergot Alkaloids Using LC-IMS-MS

Detection of Regulated Ergot Alkaloids in Food Matrices by Liquid Chromatography-Trapped Ion Mobility Spectrometry-Time-of-Flight Mass Spectrometry

Ion mobility mass spectrometry (IMS) acts as an additional separation dimension when integrated into liquid chromatography-mass spectrometry (LC-MS) workflows. LC-IMS-MS methods provide higher peak resolution, enhanced separation of isobaric and isomeric compounds, and improved signal-to-noise ratio (S/N) compared to traditional LC-MS methods. IMS provides another molecular characteristic for the identification of analytes, namely the collision cross section (CCS) parameter, reducing false positive results. Therefore, LC-IMS-MS methods address important analytical challenges in the field of food safety (i.e., detection of compounds at trace levels in complex food matrices and unambiguous identification of isobaric and isomeric molecules).
Ergot alkaloids (EAs) are a family of mycotoxins produced by fungi that attack a wide variety of grass species, including small grains such as rye, triticale, wheat, barley, millet, and oats. Maximum levels (MLs) of these mycotoxins have been established in several foodstuffs, as detailed in the Commission Regulation EC/2023/915. This new legislation includes six main EAs and their corresponding epimers, so an efficient methodology is required to properly distinguish these isomeric molecules considering their co-occurrence.
Therefore, the goal of this protocol is to show how the integration of IMS in LC-MS workflows contributes to the separation of isomeric EAs, enhancing the selectivity of the analytical method. Additionally, it illustrates how the generation of CCS libraries through the characterization of analytical standards provides higher confidence for the identification of mycotoxins. This protocol is designed to clearly explain the benefits of implementing IMS in food safety, taking as an example the determination of EAs in cereals. A QuEChERS-based extraction followed by an LC-trapped ion mobility spectrometry (TIMS)-MS analysis provided limits of quantification ranging from 0.65 to 2.6 ng/g with acceptable accuracy (although low recovery for ergotaminine) at 1.5x, 1x, and 0.5x the ML and exhibited a negligible matrix effect.

This protocol presents a validated liquid chromatography-ion mobility-high resolution mass spectrometry method to determine the presence of ergot alkaloids in food in compliance with the recently released Commission Regulation (EU) 2023/915.

Ion mobility mass spectrometry (IMS) acts as an additional separation dimension when integrated into liquid chromatography-mass spectrometry (LC-MS) ...

Tandem Mass Spectrometry

Tandem mass spectrometry is a technique that uses multiple mass analyzers in series to obtain a higher selectivity and signal-to-noise ratio for the analyte. Instruments with multiple analyzers separated by an interaction cell enable secondary fragmentation and selected study of the fragment ions.
Secondary fragmentations occur in the interaction cell and can be induced by various factors. Fragmentation induced by collision with inert gases, such as N2, Ar, He, etc., is called collision-induced dissociation (CID). Other techniques involve interaction with the surface through surface-induced dissociation (SID); interaction with low-energy electrons, called electron-capture dissociation (ECD); or through an intense laser beam, called photo-induced dissociation (PID).
Tandem mass spectrometry can be used for various experiments by varying the mode of operation of the two mass analyzers. When the first mass analyzer selects a particular precursor ion and the second mass analyzer scans all the fragmented product ions, it is called a product-ion scan, and the spectra obtained by this experiment are called product-ion spectra. Alternately, when the first mass analyzer scans all the precursor ions and the second mass analyzer selects a particular product ion, it is called &#39;precursor-ion mode&#39;, and the spectra are termed precursor-ion spectra. Another experiment can be performed by scanning both the analyzers simultaneously with an offset of a mass equal to a neutral fragment lost by the ion to be monitored. Such a mode is called &#39;neutral-loss scanning mode&#39;, and the corresponding spectra are called neutral-loss spectra.
The instrumentation of tandem mass spectrometry can be varied. Tandem-in-space spectrometers use two independent mass analyzers in two different regions in space, for example, a triple quadrupole mass spectrometer and TOF&#8211;TOF spectrometer. On the other hand, a tandem-in-time spectrometer, such as a quadrupole ion-trap instrument, uses the same spatial region to fragment the ions and dissociate and analyze a selected ion of interest, but at different times by expelling the unwanted ions. Tandem-in-time spectrometers can be used to perform MSn experiments. &#160;&#160;

Tandem mass spectrometry, or MS/MS, combines two mass analyzers in sequence, connected by a collision or interaction cell.
First, the samples are ionized ...

Education

JoVE Core

Analytical Chemistry

Unit 1

Principles of Mass Spectrometry

KEY TERMS AND CONCEPTS

Mobility Shift Affinity Capillary Electrophoresis: A Method to Analyze Sample-Ligand Interactions Depending on Differential Migration of Protein-Ligand Complexes

After preparing the method for the measurements without ligands, prepare the method for the measurements with ligands, by first using 0.1 molar EDTA solution to rinse the capillary at 2.5 bar for 1 minute. Then, use deionized water to rinse the capillary. Next, equilibrate the capillary by using ligand solution to rinse it at 2.5 bar for 1.5 minutes.
Then, inject the acetanilide solution at 0.05 bar for 6 seconds, and change the inlet and outlet vials to the ligand-containing buffer vials. Apply 0.05 bar for 2.4 seconds, in order to push the acetanilide solution further in from the tip of the capillary. Apply 10.0 kilovolts for 6 minutes, and detect the acetanilide peak at a wavelength of 200 nanometers.
After rinsing the capillary with EDTA, deionized water, and the ligand solution as before, inject the protein sample, and change the inlet and outlet vials to fresh ligand-containing buffer vials. Ultimately, repeat taking measurements with and without ligands.

Using a Cyclic Ion Mobility Spectrometer for Tandem Ion Mobility Experiments

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