Laser capture microdissection and native mass spectrometry for spatially-resolved analysis of intact protein assemblies in tissue.

Previously, we have shown that native ambient mass spectrometry imaging allows the spatial mapping of folded proteins and their complexes in thin tissue sections. Subsequent top-down native ambient mass spectrometry of adjacent tissue section enables protein identification. The challenges associated with protein identification by this approach are (i) the low abundance of proteins in tissue and associated long data acquisition timescales and (ii) irregular spatial distributions which hamper targeted sampling of the relevant tissue location. Here, we demonstrate that these challenges may be overcome through integration of laser capture microdissection in the workflow. We show identification of intact protein assemblies in rat liver tissue and apply the approach to identification of proteins in the granular layer of rat cerebellum.

High-Throughput Deconvolution of Native Protein Mass Spectrometry Imaging Data Sets for Mass Domain Analysis.

Protein mass spectrometry imaging (MSI) with electrospray-based ambient ionization techniques, such as nanospray desorption electrospray ionization (nano-DESI), generates data sets in which each pixel corresponds to a mass spectrum populated by peaks corresponding to multiply charged protein ions. Importantly, the signal associated with each protein is split among multiple charge states. These peaks can be transformed into the mass domain by spectral deconvolution. When proteins are imaged under native/non-denaturing conditions to retain non-covalent interactions, deconvolution is particularly valuable in helping interpret the data. To improve the acquisition speed, signal-to-noise ratio, and sensitivity, native MSI is usually performed using mass resolving powers that do not provide isotopic resolution, and conventional algorithms for deconvolution of lower-resolution data are not suitable for these large data sets. UniDec was originally developed to enable rapid deconvolution of complex protein mass spectra. Here, we developed an updated feature set harnessing the high-throughput module, MetaUniDec, to deconvolve each pixel of native MSI data sets and transform m/z-domain image files to the mass domain. New tools enable the reading, processing, and output of open format .imzML files for downstream analysis. Transformation of data into the mass domain also provides greater accessibility, with mass information readily interpretable by users of established protein biology tools such as sodium dodecyl sulfate polyacrylamide gel electrophoresis.

Tissue Washing Improves Native Ambient Mass Spectrometry Detection of Membrane Proteins Directly from Tissue.

Native ambient mass spectrometry enables the in situ analysis of proteins and their complexes directly from tissue, providing both structural and spatial information. Until recently, the approach was applied exclusively to the analysis of soluble proteins; however, there is a drive for new techniques that enable analysis of membrane proteins. Here we demonstrate native ambient mass spectrometry of membrane proteins, including ß-barrel and α-helical (single and multipass) integral membrane proteins and membrane-associated proteins incorporating lipid anchors, by integration of a simple washing protocol to remove soluble proteins. Mass spectrometry imaging revealed that washing did not disrupt the spatial distributions of the membrane and membrane-associated proteins. Some delocalization of the remaining soluble proteins was observed.

Liquid Extraction Surface Analysis Mass Spectrometry Imaging of Denatured Intact Proteins.

Liquid extraction surface analysis (LESA) is an ambient surface sampling technique that can be coupled with mass spectrometry (MS) to analyze analytes directly from biological substrates such as tissue sections. LESA MS involves liquid microjunction sampling of a substrate by use of a discrete volume of solvent followed by nano-electrospray ionization. As the technique makes use of electrospray ionization, it lends itself to the analysis of intact proteins. Here, we describe the use of LESA MS to analyze and image the distribution of intact denatured proteins from thin fresh frozen tissue sections.

Mass Spectrometry Detection and Imaging of a Non-Covalent Protein-Drug Complex in Tissue from Orally Dosed Rats.

Here, we demonstrate detection by mass spectrometry of an intact protein-drug complex directly from liver tissue from rats that had been orally dosed with the drug. The protein-drug complex comprised fatty acid binding protein 1, FABP1, non-covalently bound to the small molecule therapeutic bezafibrate. Moreover, we demonstrate spatial mapping of the [FABP1+bezafibrate] complex across a thin section of liver by targeted mass spectrometry imaging. This work is the first demonstration of in situ mass spectrometry analysis of a non-covalent protein-drug complex formed in vivo and has implications for early stage drug discovery by providing a route to target-drug characterization directly from the physiological environment.

Native Ambient Mass Spectrometry of an Intact Membrane Protein Assembly and Soluble Protein Assemblies Directly from Lens Tissue.

Membrane proteins constitute around two-thirds of therapeutic targets but present a significant challenge for structural analysis due to their low abundance and solubility. Existing methods for structural analysis rely on over-expression and/or purification of the membrane protein, thus removing any links back to actual physiological environment. Here, we demonstrate mass spectrometry analysis of an intact oligomeric membrane protein directly from tissue. Aquaporin-0 exists as a 113 kDa tetramer, with each subunit featuring six transmembrane helices. We report the characterisation of the intact assembly directly from a section of sheep eye lens without sample pre-treatment. Protein identity was confirmed by mass measurement of the tetramer and subunits, together with top-down mass spectrometry, and the spatial distribution was determined by mass spectrometry imaging. Our approach allows simultaneous analysis of soluble protein assemblies in the tissue.

Native Ambient Mass Spectrometry Enables Analysis of Intact Endogenous Protein Assemblies up to 145 kDa Directly from Tissue.

Untargeted label-free interrogation of proteins in their functional form directly from their physiological environment promises to transform life sciences research by providing unprecedented insight into their transient interactions with other biomolecules and xenobiotics. Native ambient mass spectrometry (NAMS) shows great potential for the structural analysis of endogenous protein assemblies directly from tissues; however, to date, this has been limited to assemblies of low molecular weight (<20 kDa) or very high abundance (hemoglobin tetramer in blood vessels, RidA homotrimer in kidney cortex tissues). The present work constitutes a step change for NAMS of protein assemblies: we demonstrate the detection and identification of a range of intact endogenous protein assemblies with various stoichiometries (dimer, trimer, and tetramer) from a range of tissue types (brain, kidney, liver) by the use of multiple NAMS techniques. Crucially, we demonstrate a greater than twofold increase in accessible molecular weight (up to 145 kDa). In addition, spatial distributions of protein assemblies up to 94 kDa were mapped in brain and kidney by nanospray desorption electrospray ionization (nano-DESI) mass spectrometry imaging.

LAP-MALDI MS coupled with machine learning: an ambient mass spectrometry approach for high-throughput diagnostics.

Large-scale population screening for early and accurate detection of disease is a key objective for future diagnostics. Ideally, diagnostic tests that achieve this goal are also cost-effective, fast and easily adaptable to new diseases with the potential of multiplexing. Mass spectrometry (MS), particularly MALDI MS profiling, has been explored for many years in disease diagnostics, most successfully in clinical microbiology but less in early detection of diseases. Here, we present liquid atmospheric pressure (LAP)-MALDI MS profiling as a rapid, large-scale and cost-effective platform for disease analysis. Using this new platform, two different types of tests exemplify its potential in early disease diagnosis and response to therapy. First, it is shown that LAP-MALDI MS profiling detects bovine mastitis two days before its clinical manifestation with a sensitivity of up to 70% and a specificity of up to 100%. This highly accurate, pre-symptomatic detection is demonstrated by using a large set of milk samples collected weekly over six months from approximately 500 dairy cows. Second, the potential of LAP-MALDI MS in antimicrobial resistance (AMR) detection is shown by employing the same mass spectrometric setup and similarly simple sample preparation as for the early detection of mastitis.

Native Ambient Mass Spectrometry Imaging of Ligand-Bound and Metal-Bound Proteins in Rat Brain.

Label-free spatial mapping of the noncovalent interactions of proteins in their tissue environment has the potential to revolutionize life sciences research by providing opportunities for the interrogation of disease progression, drug interactions, and structural and molecular biology more broadly. Here, we demonstrate mass spectrometry imaging of endogenous intact noncovalent protein-ligand complexes in rat brain. The spatial distributions of a range of ligand-bound and metal-bound proteins were mapped in thin tissue sections by use of nanospray-desorption electrospray ionization. Proteins were identified directly from the tissue by top-down mass spectrometry. Three GDP-binding proteins (ADP ribosylation factor ARF3, ARF1, and GTPase Ran) were detected, identified, and imaged in their ligand-bound form. The nature of the ligand was confirmed by multiple rounds of tandem mass spectrometry. In addition, the metal-binding proteins parvalbumin-α and carbonic anhydrase 2 were detected, identified, and imaged in their native form, i.e., parvalbumin-α + 2Ca2+ and carbonic anhydrase + Zn2+. GTPase Ran was detected with both GDP and Mg2+ bound. Several natively monomeric proteins displaying distinct spatial distributions were also identified by top-down mass spectrometry. Protein mass spectrometry imaging was achieved at a spatial resolution of 200 µm.

Native Ambient Mass Spectrometry of an Intact Membrane Protein Assembly and Soluble Protein Assemblies Directly from Lens Tissue.

Membrane proteins constitute around two-thirds of therapeutic targets but present a significant challenge for structural analysis due to their low abundance and solubility. Existing methods for structural analysis rely on over-expression and/or purification of the membrane protein, thus removing any links back to actual physiological environment. Here, we demonstrate mass spectrometry analysis of an intact oligomeric membrane protein directly from tissue. Aquaporin-0 exists as a 113 kDa tetramer, with each subunit featuring six transmembrane helices. We report the characterisation of the intact assembly directly from a section of sheep eye lens without sample pre-treatment. Protein identity was confirmed by mass measurement of the tetramer and subunits, together with top-down mass spectrometry, and the spatial distribution was determined by mass spectrometry imaging. Our approach allows simultaneous analysis of soluble protein assemblies in the tissue.

Mass Spectrometry Detection and Imaging of a Non-Covalent Protein-Drug Complex in Tissue from Orally Dosed Rats.

Here, we demonstrate detection by mass spectrometry of an intact protein-drug complex directly from liver tissue from rats that had been orally dosed with the drug. The protein-drug complex comprised fatty acid binding protein 1, FABP1, non-covalently bound to the small molecule therapeutic bezafibrate. Moreover, we demonstrate spatial mapping of the [FABP1+bezafibrate] complex across a thin section of liver by targeted mass spectrometry imaging. This work is the first demonstration of in situ mass spectrometry analysis of a non-covalent protein-drug complex formed in vivo and has implications for early stage drug discovery by providing a route to target-drug characterization directly from the physiological environment.

Native Mass Spectrometry Imaging of Proteins and Protein Complexes by Nano-DESI.

Previously, we have demonstrated native mass spectrometry imaging (native MSI) in which the spatial distribution of proteins maintained in their native-like, folded conformations was determined using liquid extraction surface analysis (LESA). While providing an excellent testbed for proof of principle, the spatial resolution of LESA is currently limited for imaging primarily by the physical size of the sampling pipette tip. Here, we report the adoption of nanospray-desorption electrospray ionization (nano-DESI) for native MSI, delivering substantial improvements in resolution versus native LESA MSI. In addition, native nano-DESI may be used for location-targeted top-down proteomics analysis directly from tissue. Proteins, including a homodimeric complex not previously detected by native MSI, were identified through a combination of collisional activation, high-resolution MS and proton transfer charge reduction.

Speciation and milk adulteration analysis by rapid ambient liquid MALDI mass spectrometry profiling using machine learning.

Growing interest in food quality and traceability by regulators as well as consumers demands advances in more rapid, versatile and cost-effective analytical methods. Milk, as most food matrices, is a heterogeneous mixture composed of metabolites, lipids and proteins. One of the major challenges is to have simultaneous, quantitative detection (profiling) of this panel of biomolecules to gather valuable information for assessing food quality, traceability and safety. Here, for milk analysis, atmospheric pressure matrix-assisted laser desorption/ionization employing homogenous liquid sample droplets was used on a Q-TOF mass analyzer. This method has the capability to produce multiply charged proteinaceous ions as well as highly informative profiles of singly charged lipids/metabolites. In two examples, this method is coupled with user-friendly machine-learning software. First, rapid speciation of milk (cow, goat, sheep and camel) is demonstrated with 100% classification accuracy. Second, the detection of cow milk as adulterant in goat milk is shown at concentrations as low as 5% with 92.5% sensitivity and 94.5% specificity.

Production and analysis of multiply charged negative ions by liquid atmospheric pressure matrix-assisted laser desorption/ionization mass spectrometry.

RATIONALE: Liquid atmospheric pressure matrix-assisted laser desorption/ionisation (AP-MALDI) has been shown to enable the production of electrospray ionisation (ESI)-like multiply charged analyte ions with little sample consumption and long-lasting, robust ion yield for sensitive analysis by mass spectrometry (MS). Previous reports have focused on positive ion production. Here, we report an initial optimisation of liquid AP-MALDI for ESI-like negative ion production and its application to the analysis of peptides/proteins, DNA and lipids. METHODS: The instrumentation employed for this study is identical to that of earlier liquid AP-MALDI MS studies for positive analyte ion production with a simple non-commercial AP ion source that is attached to a Waters Synapt G2-Si mass spectrometer and incorporates a heated ion transfer tube. The preparation of liquid MALDI matrices is similar to positive ion mode analysis but has been adjusted for negative ion mode by changing the chromophore to 3-aminoquinoline and 9-aminoacridine for further improvements. RESULTS: For DNA, liquid AP-MALDI MS analysis benefited from switching to 9-aminoacridine-based MALDI samples and the negative ion mode, increasing the number of charges by up to a factor of 2 and the analyte ion signal intensities by more than 10-fold compared with the positive ion mode. The limit of detection was recorded at around 10 fmol for ATGCAT. For lipids, negative ion mode analysis provided a fully orthogonal set of detected lipids. CONCLUSIONS: Negative ion mode is a sensitive alternative to positive ion mode in liquid AP-MALDI MS analysis. In particular, the analysis of lipids and DNA benefited from the complementarity of the detected lipid species and the vastly greater DNA ion signal intensities in negative ion mode.

Native Mass Spectrometry Imaging and In Situ Top-Down Identification of Intact Proteins Directly from Tissue.

Mass spectrometry imaging (MSI) provides information on the spatial distribution of molecules within a biological substrate without the requirement for labeling. Its broad specificity, i.e., the capability to spatially profile any analyte ion detected, constitutes a major advantage over other imaging techniques. A separate branch of mass spectrometry, native mass spectrometry, provides information relating to protein structure through retention of solution-phase interactions in the gas phase. Integration of MSI and native mass spectrometry ("native MSI") affords opportunities for simultaneous acquisition of spatial and structural information on proteins directly from their physiological environment. Here, we demonstrate significant improvements in native MSI and associated protein identification of intact proteins and protein assemblies in thin sections of rat kidney by use of liquid extraction surface analysis on a state-of-the-art Orbitrap mass spectrometer optimized for intact protein analysis. Proteins of up to 47 kDa, including a trimeric protein complex, were imaged and identified.

High-Field Asymmetric Waveform Ion Mobility Spectrometry and Native Mass Spectrometry: Analysis of Intact Protein Assemblies and Protein Complexes.

High-field asymmetric waveform ion mobility spectrometry (FAIMS) enables the separation of ions on the basis of their differential mobility in an asymmetric oscillating electric field. We, and others, have previously demonstrated the benefits of FAIMS for the analysis of peptides and denatured proteins. To date, FAIMS has not been integrated with native mass spectrometry of folded proteins and protein complexes, largely due to concerns over the heating effects associated with the high electric fields employed. Here, we demonstrate the newly introduced cylindrical FAIMS Pro device coupled with an Orbitrap Eclipse enables analysis of intact protein assemblies up to 147 kDa. No evidence for dissociation was detected suggesting that any field heating is insufficient to disrupt the noncovalent interactions governing these assemblies. Moreover, the FAIMS device was integrated into native liquid extraction surface analysis (LESA) MS of protein assemblies directly from thin tissue sections. Intact tetrameric hemoglobin (64 kDa) and trimeric reactive intermediate deiminase A (RidA, 43 kDa) were detected. Improvements in signal-to-noise of between 1.5× and 12× were observed for these protein assemblies on integration of FAIMS.

Native LESA TWIMS-MSI: Spatial, Conformational, and Mass Analysis of Proteins and Protein Complexes.

We have previously demonstrated native liquid extraction surface analysis (LESA) mass spectrometry imaging of small intact proteins in thin tissue sections. We also showed calculation of collision cross sections for specific proteins extracted from discrete locations in tissue by LESA traveling wave ion mobility spectrometry (TWIMS). Here, we demonstrate an integrated native LESA TWIMS mass spectrometry imaging (MSI) workflow, in which ion mobility separation is central to the imaging experiment and which provides spatial, conformational, and mass information on endogenous proteins in a single experiment. The approach was applied to MSI of a thin tissue section of mouse kidney. The results show that the benefits of integration of TWIMS include improved specificity of the ion images and the capacity to calculate collision cross sections for any protein or protein complex detected in any pixel (without a priori knowledge of the presence of the protein).

In situ mass spectrometry analysis of intact proteins and protein complexes from biological substrates.

Advances in sample preparation, ion sources and mass spectrometer technology have enabled the detection and characterisation of intact proteins. The challenges associated include an appropriately soft ionisation event, efficient transmission and detection of the often delicate macromolecules. Ambient ion sources, in particular, offer a wealth of strategies for analysis of proteins from solution environments, and directly from biological substrates. The last two decades have seen rapid development in this area. Innovations include liquid extraction surface analysis, desorption electrospray ionisation and nanospray desorption electrospray ionisation. Similarly, developments in native mass spectrometry allow protein-protein and protein-ligand complexes to be ionised and analysed. Identification and characterisation of these large ions involves a suite of hyphenated mass spectrometry techniques, often including the coupling of ion mobility spectrometry and fragmentation techniques. The latter include collision, electron and photon-induced methods, each with their own characteristics and benefits for intact protein identification. In this review, recent developments for in situ protein analysis are explored, with a focus on ion sources and tandem mass spectrometry techniques used for identification.

Atmospheric Pressure Ultraviolet Laser Desorption and Ionization from Liquid Samples for Native Mass Spectrometry.

Understanding protein structure is vital for evaluating protein interactions with drugs, proteins, and other ligands. Native mass spectrometry (MS) is proving to be invaluable for this purpose, enabling analysis of "native-like" samples that mimic physiological conditions. Native MS is usually performed by electrospray ionization (ESI) with its soft ionization processes and the generation of multiply charged ions proving favorable for conformation retention and high mass analysis, respectively. There is scope to expand the currently available toolset, specifically to other soft ionization techniques such as soft laser desorption, for applications in areas like high-throughput screening and MS imaging. In this Letter, observations made from native MS experiments using an ultraviolet (UV) laser-based ion source operating at atmospheric pressure are described. The ion source is capable of producing predominately multiply charged ions similar to ESI. Proteins and protein complexes were analyzed from a native-like sample droplet to investigate the technique. Ion mobility-mass spectrometry (IM-MS) measurements showed that folded protein conformations were detected for ions with low charge states. This observation indicates the source is suitable for native MS analysis and should be further developed for higher mass analysis in the future.

Liquid Atmospheric Pressure Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Adds Enhanced Functionalities to MALDI MS Profiling for Disease Diagnostics.

A liquid matrix-assisted laser desorption/ionization (liquid MALDI) method has been developed for high-throughput atmospheric pressure (AP) mass spectrometry (MS) analysis of the molecular content of crude bioliquids for disease diagnostics. The presented method is rapid and highly robust, enabling its application in environments where speed and low-cost high-throughput analyses are required. Importantly, because of the creation of multiply charged analyte ions, it provides additional functionalities that conventional solid MALDI MS profiling is lacking, including the use of high-performance mass analyzers with limited m/z range. The concomitant superior MS/MS performance that is achieved similar to ESI MS/MS adds greater analytical power and specificity to MALDI MS profiling while retaining the advantages of a fast laser-based analysis system and off-line large-scale sample preparation. The potential of this MALDI MS profiling method is demonstrated on the detection of dairy cow mastitis, which is a substantial economic burden on the dairy industry with losses of hundreds of dollars per diseased cow per year, equating to a total annual loss of billions of dollars, as well as leading to the use of large quantities of antibiotics, adding to the proliferation of antimicrobial resistance. Only small amounts of aliquots obtained from the daily farm milking process were prepared for liquid MALDI MS profiling using a simple one-pot/two-step analyte extraction. Automated analysis was performed using a custom-built AP-MALDI ion source, enabling the simultaneous detection of lipids, peptides, and proteins. Diagnostic, multiply charged, proteinaceous ions were easily sequenced and identified by MS/MS experiments. Samples were classified according to mastitis status using multivariate analysis, achieving 98.5% accuracy (100% specificity) determined by "leave 20% out" cross-validation. The methodology is generally applicable to AP-MALDI MS profiling on most commercial high-resolution mass spectrometers, with the potential for expansion into hospitals for rapid assessment of human and other biofluids.