Harmonization of Rapid Evaporative Ionization Mass Spectrometry Workflows across Four Sites and Testing Using Reference Material and Local Food-Grade Meats.

Rapid evaporative ionization mass spectrometry (REIMS) is a direct tissue metabolic profiling technique used to accurately classify tissues using pre-built mass spectral databases. The reproducibility of the analytical equipment, methodology and tissue classification algorithms has yet to be evaluated over multiple sites, which is an essential step for developing this technique for future clinical applications. In this study, we harmonized REIMS methodology using single-source reference material across four sites with identical equipment: Imperial College London (UK); Waters Research Centre (Hungary); Maastricht University (The Netherlands); and Queen's University (Canada). We observed that method harmonization resulted in reduced spectral variability across sites. Each site then analyzed four different types of locally-sourced food-grade animal tissue. Tissue recognition models were created at each site using multivariate statistical analysis based on the different metabolic profiles observed in the m/z range of 600-1000, and these models were tested against data obtained at the other sites. Cross-validation by site resulted in 100% correct classification of two reference tissues and 69-100% correct classification for food-grade meat samples. While we were able to successfully minimize between-site variability in REIMS signals, differences in animal tissue from local sources led to significant variability in the accuracy of an individual site's model. Our results inform future multi-site REIMS studies applied to clinical samples and emphasize the importance of carefully-annotated samples that encompass sufficient population diversity.

Matrix Assisted Rapid Evaporative Ionization Mass Spectrometry.

Rapid evaporative ionization mass spectrometry (REIMS) is a highly versatile technique allowing the sampling of a range of biological solid or liquid samples with no sample preparation. The cost of such a direct approach is that certain sample types provide only moderate amounts of chemical information. Here, we introduce a matrix assisted version of the technique (MA-REIMS), where an aerosol of a pure solvent, such as isopropanol, is mixed with the sample aerosol generated by rapid evaporation of the sample, and it is shown to enhance the signal intensity obtained from a REIMS sampling event by over 2 orders of magnitude. Such an increase greatly expands the scope of the technique, while providing additional benefits such as reducing the fouling of the REIMS source and allowing for a simple method of constant introduction of a calibration correction compound for accurate mass measurements. A range of experiments are presented in order to investigate the processes that occur within this modified approach, and applications where such enhancements are critical, such as intrasurgical tissue identification, are discussed.

Acoustic Mist Ionization Platform for Direct and Contactless Ultrahigh-Throughput Mass Spectrometry Analysis of Liquid Samples.

Mass spectrometry (MS) has many advantages as a quantitative detection technology for applications within drug discovery. However, current methods of liquid sample introduction to a detector are slow and limit the use of mass spectrometry for kinetic and high-throughput applications. We present the development of an acoustic mist ionization (AMI) interface capable of contactless nanoliter-scale "infusion" of up to three individual samples per second into the mass detector. Installing simple plate handling automation allowed us to reach a throughput of 100 000 samples per day on a single mass spectrometer. We applied AMI-MS to identify inhibitors of a human histone deacetylase from AstraZeneca's collection of 2 million small molecules and measured their half-maximal inhibitory concentration. The speed, sensitivity, simplicity, robustness, and consumption of nanoliter volumes of sample suggest that this technology will have a major impact across many areas of basic and applied research.

Optimised Desorption Electrospray Ionisation Mass Spectrometry Imaging (DESI-MSI) for the Analysis of Proteins/Peptides Directly from Tissue Sections on a Travelling Wave Ion Mobility Q-ToF.

Desorption electrospray ionisation mass spectrometry imaging (DESI-MSI) is typically known for the ionisation of small molecules such as lipids and metabolites, in singly charged form. Here we present a method that allows the direct detection of proteins and peptides in multiply charged forms directly from tissue sections by DESI. Utilising a heated mass spectrometer inlet capillary, combined with ion mobility separation (IMS), the conditions with regard to solvent composition, nebulising gas flow, and solvent flow rate have been explored and optimised. Without the use of ion mobility separation prior to mass spectrometry analysis, only the most abundant charge series were observed. In addition to the dominant haemoglobin subunit(s) related trend line in the m/z vs drift time (DT) 2D plot, trend lines were found relating to background solvent peaks, residual lipids and, more importantly, small proteins/large peptides of lower abundance. These small proteins/peptides were observed with charge states from 1+ to 12+, the majority of which could only be resolved from the background when using IMS. By extracting charge series from the 2D m/z vs DT plot, a number of proteins could be tentatively assigned by accurate mass. Tissue images were acquired with a pixel size of 150 µm showing a marked improvement in protein image resolution compared to other liquid-based ambient imaging techniques such as liquid extraction surface analysis (LESA) and continuous-flow liquid microjunction surface sampling probe (LMJ-SSP) imaging. Graphical Abstract ᅟ.

A real time metabolomic profiling approach to detecting fish fraud using rapid evaporative ionisation mass spectrometry.

INTRODUCTION: Fish fraud detection is mainly carried out using a genomic profiling approach requiring long and complex sample preparations and assay running times. Rapid evaporative ionisation mass spectrometry (REIMS) can circumvent these issues without sacrificing a loss in the quality of results. OBJECTIVES: To demonstrate that REIMS can be used as a fast profiling technique capable of achieving accurate species identification without the need for any sample preparation. Additionally, we wanted to demonstrate that other aspects of fish fraud other than speciation are detectable using REIMS. METHODS: 478 samples of five different white fish species were subjected to REIMS analysis using an electrosurgical knife. Each sample was cut 8-12 times with each one lasting 3-5 s and chemometric models were generated based on the mass range m/z 600-950 of each sample. RESULTS: The identification of 99 validation samples provided a 98.99% correct classification in which species identification was obtained near-instantaneously (≈ 2 s) unlike any other form of food fraud analysis. Significant time comparisons between REIMS and polymerase chain reaction (PCR) were observed when analysing 6 mislabelled samples demonstrating how REIMS can be used as a complimentary technique to detect fish fraud. Additionally, we have demonstrated that the catch method of fish products is capable of detection using REIMS, a concept never previously reported. CONCLUSIONS: REIMS has been proven to be an innovative technique to help aid the detection of fish fraud and has the potential to be utilised by fisheries to conduct their own quality control (QC) checks for fast accurate results.

Faster, More Reproducible DESI-MS for Biological Tissue Imaging.

A new, more robust sprayer for desorption electrospray ionization (DESI) mass spectrometry imaging is presented. The main source of variability in DESI is thought to be the uncontrolled variability of various geometric parameters of the sprayer, primarily the position of the solvent capillary, or more specifically, its positioning within the gas capillary or nozzle. If the solvent capillary is off-center, the sprayer becomes asymmetrical, making the geometry difficult to control and compromising reproducibility. If the stiffness, tip quality, and positioning of the capillary are improved, sprayer reproducibility can be improved by an order of magnitude. The quality of the improved sprayer and its potential for high spatial resolution imaging are demonstrated on human colorectal tissue samples by acquisition of images at pixel sizes of 100, 50, and 20 µm, which corresponds to a lateral resolution of 40-60 µm, similar to the best values published in the literature. The high sensitivity of the sprayer also allows combination with a fast scanning quadrupole time-of-flight mass spectrometer. This provides up to 30 times faster DESI acquisition, reducing the overall acquisition time for a 10 mm × 10 mm rat brain sample to approximately 1 h. Although some spectral information is lost with increasing analysis speed, the resulting data can still be used to classify tissue types on the basis of a previously constructed model. This is particularly interesting for clinical applications, where fast, reliable diagnosis is required. Graphical Abstract ᅟ.

DESI Mass Spectrometry Imaging (MSI).

Desorption Electrospray Ionization (DESI) mass spectrometry is a technique that allows chemical information to be obtained directly from a wide range of surfaces. Using a 2D stage, DESI can be implemented in an imaging mode whereby MS spectra are collected by rastering the spray across the whole surface. Here, we describe the implementation and optimization of DESI imaging for metabolites and lipids from tissue sections using oa-TOF mass spectrometers.

Identification of the Species of Origin for Meat Products by Rapid Evaporative Ionization Mass Spectrometry.

Increasingly abundant food fraud cases have brought food authenticity and safety into major focus. This study presents a fast and effective way to identify meat products using rapid evaporative ionization mass spectrometry (REIMS). The experimental setup was demonstrated to be able to record a mass spectrometric profile of meat specimens in a time frame of <5 s. A multivariate statistical algorithm was developed and successfully tested for the identification of animal tissue with different anatomical origin, breed, and species with 100% accuracy at species and 97% accuracy at breed level. Detection of the presence of meat originating from a different species (horse, cattle, and venison) has also been demonstrated with high accuracy using mixed patties with a 5% detection limit. REIMS technology was found to be a promising tool in food safety applications providing a reliable and simple method for the rapid characterization of food products.

Rapid evaporative ionization mass spectrometry imaging platform for direct mapping from bulk tissue and bacterial growth media.

Rapid evaporative ionization mass spectrometry (REIMS) technology allows real time intraoperative tissue classification and the characterization and identification of microorganisms. In order to create spectral libraries for training the classification models, reference data need to be acquired in large quantities as classification accuracy generally improves as a function of number of training samples. In this study, we present an automated high-throughput method for collecting REIMS data from heterogeneous organic tissue. The underlying instrumentation consists of a 2D stage with an additional high-precision z-axis actuator that is equipped with an electrosurgical diathermy-based sampling probe. The approach was validated using samples of human liver with metastases and bacterial strains, cultured on solid medium, belonging to the species P. aeruginosa, B. subtilis, and S. aureus. For both sample types, spatially resolved spectral information was obtained that resulted in clearly distinguishable multivariate clustering between the healthy/cancerous liver tissues and between the bacterial species.

Changes in protein structure monitored by use of gas-phase hydrogen/deuterium exchange.

The study of protein conformation by solution-phase hydrogen/deuterium exchange (HDX) coupled to MS is well documented. This involves monitoring the exchange of backbone amide protons with deuterium and provides details concerning the protein's tertiary structure. However, undesired back-exchange during post-HDX analyses can be difficult to control. Here, gas-phase HDX-MS, during which labile hydrogens on amino acid side chains are exchanged in sub-millisecond time scales, has been employed to probe changes within protein structures. Addition of the solvent 2,2,2-trifluoroethanol to a protein in solution can affect the structure of the protein, resulting in an increase in secondary and/or tertiary structure which is detected using circular dichroism. Using a Synapt G2-S ESI-mass spectrometer modified to allow deuterated ammonia into the transfer ion guide (situated between the ion mobility cell and the TOF analyser), gas-phase HDX-MS is shown to reflect minor structural changes experienced by the proteins ß-lactoglobulin and ubiquitin, as observed by the reduction in the level of deuterium incorporation. Additionally, the use of gas-phase HDX-MS to distinguish between co-populated proteins conformers within a solution is demonstrated with the disordered protein calmodulin; the gas-phase HDX-MS results correspond directly with complementary data obtained by use of ion mobility spectrometry-MS.

Site-specific analysis of gas-phase hydrogen/deuterium exchange of peptides and proteins by electron transfer dissociation.

To interpret the wealth of information contained in the hydrogen/deuterium exchange (HDX) behavior of peptides and proteins in the gas-phase, analytical tools are needed to resolve the HDX of individual exchanging sites. Here we show that ETD can be combined with fast gas-phase HDX in ND(3) gas and used to monitor the exchange of side-chain hydrogens of individual residues in both small peptide ions and larger protein ions a few milliseconds after electrospray. By employing consecutive traveling wave ion guides in a mass spectrometer, peptide and protein ions were labeled on-the-fly (0.1-10 ms) in ND(3) gas and subsequently fragmented by ETD. Fragment ions were separated using ion mobility and mass analysis enabled the determination of the gas-phase deuterium uptake of individual side-chain sites in a range of model peptides of different size and sequence as well as two proteins; cytochrome C and ubiquitin. Gas-phase HDX-ETD experiments on ubiquitin ions ionized from both denaturing and native solution conditions suggest that residue-specific HDX of side-chain hydrogens is sensitive to secondary and tertiary structural features occurring in both near-native and unfolded gas-phase conformers present shortly after electrospray. The described approach for online gas-phase HDX and ETD paves the way for making mass spectrometry techniques based on gas-phase HDX more applicable in bioanalytical research.

ETD in a traveling wave ion guide at tuned Z-spray ion source conditions allows for site-specific hydrogen/deuterium exchange measurements.

The recent application of electron transfer dissociation (ETD) to measure the hydrogen exchange of proteins in solution at single-residue resolution (HX-ETD) paves the way for mass spectrometry-based analyses of biomolecular structure at an unprecedented level of detail. The approach requires that activation of polypeptide ions prior to ETD is minimal so as to prevent undesirable gas-phase randomization of the deuterium label from solution (i.e., hydrogen scrambling). Here we explore the use of ETD in a traveling wave ion guide of a quadrupole-time-of-flight (Q-TOF) mass spectrometer with a "Z-spray" type ion source, to measure the deuterium content of individual residues in peptides. We systematically identify key parameters of the Z-spray ion source that contribute to collisional activation and define conditions that allow ETD experiments to be performed in the traveling wave ion guide without gas-phase hydrogen scrambling. We show that ETD and supplemental collisional activation in a subsequent traveling wave ion guide allows for improved extraction of residue-specific deuterium contents in peptides with low charge. Our results demonstrate the feasibility, and illustrate the advantages of performing HX-ETD experiments on a high-resolution Q-TOF instrument equipped with traveling wave ion guides. Determination of parameters of the Z-spray ion source that contribute to ion heating are similarly pertinent to a growing number of MS applications that also rely on an energetically gentle transfer of ions into the gas-phase, such as the analysis of biomolecular structure by native mass spectrometry in combination with gas-phase ion-ion/ion-neutral reactions or ion mobility spectrometry.

Gas-phase hydrogen/deuterium exchange in a traveling wave ion guide for the examination of protein conformations.

Accumulating evidence suggests that solution-phase conformations of small globular proteins and large molecular protein assemblies can be preserved for milliseconds after electrospray ionization. Thus, the study of proteins in the gas phase on this time scale is highly desirable. Here we demonstrate that a traveling wave ion guide (TWIG) of a Synapt mass spectrometer offers a highly suitable environment for rapid and efficient gas-phase hydrogen/deuterium exchange (HDX). Gaseous ND(3) was introduced into either the source TWIG or the TWIG located just after the ion mobility cell, such that ions underwent HDX as they passed through the ND(3) on the way to the time-of-flight analyzer. The extent of deuterium labeling could be controlled by varying the quantity of ND(3) or the speed of the traveling wave. The gas-phase HDX of model peptides corresponded to labeling of primarily fast exchanging sites due to the short labeling times (ranging from 0.1 to 10 ms). In addition to peptides, gas-phase HDX of ubiquitin, cytochrome c, lysozyme, and apomyoglobin were examined. We conclude that HDX of protein ions in a TWIG is highly sensitive to protein conformation, enables the detection of conformers present on submilliseconds time scales, and can readily be combined with ion mobility spectrometry.

Applications of a travelling wave-based radio-frequency-only stacked ring ion guide.

The use of radio-frequency (RF)-only ion guides for efficient transport of ions through regions of a mass spectrometer where the background gas pressure is relatively high is widespread in present instrumentation. Whilst multiple collisions between ions and the background gas can be beneficial, for example in inducing fragmentation and/or decreasing the spread in ion energies, the resultant reduction of ion axial velocity can be detrimental in modes of operation where a rapidly changing influx of ions to the gas-filled ion guide needs to be reproduced at the exit. In general, the RF-only ion guides presently in use are based on multipole rod sets. Here we report investigations into a new mode of ion propulsion within an RF ion guide based on a stack of ring electrodes. Ion propulsion is produced by superimposing a voltage pulse on the confining RF of an electrode and then moving the pulse to an adjacent electrode and so on along the guide to provide a travelling voltage wave on which the ions can surf. Through appropriate choice of the travelling wave pulse height, velocity and gas pressure it will be shown that the stacked ring ion guide with the travelling wave is effective as a collision cell in a tandem mass spectrometer where fast mass scanning or switching is required, as an ion mobility separator at pressures around 0.2 mbar, as an ion delivery device for enhancement of duty cycle on an orthogonal acceleration time-of-flight (oa-TOF) mass analyser, and as an ion fragmentation device at higher wave velocities.