Advances in mass spectrometry-enabled multiomics at single-cell resolution.

Biological organisms are multifaceted, intricate systems where slight perturbations can result in extensive changes in gene expression, protein abundance and/or activity, and metabolic flux. These changes occur at different timescales, spatially across cells of heterogeneous origins, and within single-cells. Hence, multimodal measurements at the smallest biological scales are necessary to capture dynamic changes in heterogeneous biological systems. Of the analytical techniques used to measure biomolecules, mass spectrometry (MS) has proven to be a powerful option due to its sensitivity, robustness, and flexibility with regard to the breadth of biomolecules that can be analyzed. Recently, many studies have coupled MS to other analytical techniques with the goal of measuring multiple modalities from the same single-cell. It is with these concepts in mind that we focus this review on MS-enabled multiomic measurements at single-cell or near-single- cell resolution.

Spatial top-down proteomics for the functional characterization of human kidney.

Background: The Human Proteome Project has credibly detected nearly 93% of the roughly 20,000 proteins which are predicted by the human genome. However, the proteome is enigmatic, where alterations in amino acid sequences from polymorphisms and alternative splicing, errors in translation, and post-translational modifications result in a proteome depth estimated at several million unique proteoforms. Recently mass spectrometry has been demonstrated in several landmark efforts mapping the human proteoform landscape in bulk analyses. Herein, we developed an integrated workflow for characterizing proteoforms from human tissue in a spatially resolved manner by coupling laser capture microdissection, nanoliter-scale sample preparation, and mass spectrometry imaging. Results: Using healthy human kidney sections as the case study, we focused our analyses on the major functional tissue units including glomeruli, tubules, and medullary rays. After laser capture microdissection, these isolated functional tissue units were processed with microPOTS (microdroplet processing in one-pot for trace samples) for sensitive top-down proteomics measurement. This provided a quantitative database of 616 proteoforms that was further leveraged as a library for mass spectrometry imaging with near-cellular spatial resolution over the entire section. Notably, several mitochondrial proteoforms were found to be differentially abundant between glomeruli and convoluted tubules, and further spatial contextualization was provided by mass spectrometry imaging confirming unique differences identified by microPOTS, and further expanding the field-of-view for unique distributions such as enhanced abundance of a truncated form (1-74) of ubiquitin within cortical regions. Conclusions: We developed an integrated workflow to directly identify proteoforms and reveal their spatial distributions. Where of the 20 differentially abundant proteoforms identified as discriminate between tubules and glomeruli by microPOTS, the vast majority of tubular proteoforms were of mitochondrial origin (8 of 10) where discriminate proteoforms in glomeruli were primarily hemoglobin subunits (9 of 10). These trends were also identified within ion images demonstrating spatially resolved characterization of proteoforms that has the potential to reshape discovery-based proteomics because the proteoforms are the ultimate effector of cellular functions. Applications of this technology have the potential to unravel etiology and pathophysiology of disease states, informing on biologically active proteoforms, which remodel the proteomic landscape in chronic and acute disorders.

Mass spectrometry imaging of natural carbonyl products directly from agar-based microbial interactions using 4-APEBA derivatization.

Aliphatic carboxylic acids, aldehydes, and ketones play diverse roles in microbial adaptation to their microenvironment, from excretion as toxins to adaptive metabolites for membrane fluidity. However, the spatial distribution of these molecules throughout biofilms and how microbes in these environments exchange these molecules remain elusive for many of these bioactive species due to inefficient molecular imaging strategies. Herein, we apply on-tissue chemical derivatization (OTCD) using 4-(2-((4-bromophenethyl)dimethylammonio)ethoxy)benzenaminium dibromide (4-APEBA) on a co-culture of a soil bacterium (Bacillus subtilis NCIB 3610) and fungus (Fusarium sp. DS 682) grown on agar as our model system. Using matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI), we spatially resolved more than 300 different metabolites containing carbonyl groups within this model system. Various spatial patterns are observable in these species, which indicate possible extracellular or intercellular processes of the metabolites and their up- or downregulation during microbial interaction. The unique chemistry of our approach allowed us to bring additional confidence in accurate carbonyl identification, especially when multiple isomeric candidates were possible, and this provided the ability to generate hypotheses about the potential role of some aliphatic carbonyls in this B. subtilis/Fusarium sp. interaction. The results shown here demonstrate the utility of 4-ABEBA-based OTCD MALDI-MSI in probing interkingdom interactions directly from microbial co-cultures, and these methods will enable future microbial interaction studies with expanded metabolic coverage.IMPORTANCEThe metabolic profiles within microbial biofilms and interkingdom interactions are extremely complex and serve a variety of functions, which include promoting colonization, growth, and survival within competitive and symbiotic environments. However, measuring and differentiating many of these molecules, especially in an in situ fashion, remains a significant analytical challenge. We demonstrate a chemical derivatization strategy that enabled highly sensitive, multiplexed mass spectrometry imaging of over 300 metabolites from a model microbial co-culture. Notably, this approach afforded us to visualize over two dozen classes of ketone-, aldehyde-, and carboxyl-containing molecules, which were previously undetectable from colonies grown on agar. We also demonstrate that this chemical derivatization strategy can enable the discrimination of isobaric and isomeric metabolites without the need for orthogonal separation (e.g., online chromatography or ion mobility). We anticipate that this approach will further enhance our knowledge of metabolic regulation within microbiomes and microbial systems used in bioengineering applications.

Expanded Coverage of Phytocompounds by Mass Spectrometry Imaging Using On-Tissue Chemical Derivatization by 4-APEBA.

Probing the entirety of any species metabolome is an analytical grand challenge, especially on a cellular scale. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) is a common spatial metabolomics assay, but this technique has limited molecular coverage for several reasons. To expand the application space of spatial metabolomics, we developed an on-tissue chemical derivatization (OTCD) workflow using 4-APEBA for the confident identification of several dozen elusive phytocompounds. Overall, this new OTCD method enabled the annotation of roughly 280 metabolites, with only a 10% overlap in metabolic coverage when compared to analog negative ion mode MALDI-MSI on serial sections. We demonstrate that 4-APEBA outperforms other derivatization agents by providing: (1) broad specificity toward carbonyls, (2) low background, and (3) introduction of bromine isotopes. Notably, the latter two attributes also facilitate more confidence in our bioinformatics for data processing. The workflow detailed here trailblazes a path toward spatial hormonomics within plant samples, enhancing the detection of carboxylates, aldehydes, and plausibly other carbonyls. As such, several phytohormones, which have various roles within stress responses and cellular communication, can now be spatially profiled, as demonstrated in poplar root and soybean root nodule.

IsoMatchMS: Open-Source Software for Automated Annotation and Visualization of High Resolution MALDI-MS Spectra.

Due to its speed, accuracy, and adaptability to various sample types, matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has become a popular method to identify molecular isotope profiles from biological samples. Often MALDI-MS data do not include tandem MS fragmentation data, and thus the identification of compounds in samples requires external databases so that the accurate mass of detected signals can be matched to known molecular compounds. Most relevant MALDI-MS software tools developed to confirm compound identifications are focused on small molecules (e.g., metabolites, lipids) and cannot be easily adapted to protein data due to their more complex isotopic distributions. Here, we present an R package called IsoMatchMS for the automated annotation of MALDI-MS data for multiple datatypes: intact proteins, peptides, and glycans. This tool accepts already derived molecular formulas or, for proteomics applications, can derive molecular formulas from a list of input peptides or proteins including proteins with post-translational modifications. Visualization of all matched isotopic profiles is provided in a highly accessible HTML format called a trelliscope display, which allows users to filter and sort by several parameters such as match scores and the number of peaks matched. IsoMatchMS simplifies the annotation and visualization of MALDI-MS data for downstream analyses.

Experimental Assessment of Mammalian Lipidome Complexity Using Multimodal 21 T FTICR Mass Spectrometry Imaging.

Herein, we assess the complementarity and complexity of data that can be detected within mammalian lipidome mass spectrometry imaging (MSI) via matrix-assisted laser desorption ionization (MALDI) and nanospray desorption electrospray ionization (nano-DESI). We do so by employing 21 T Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) with absorption mode FT processing in both cases, allowing unmatched mass resolving power per unit time (≥613k at m/z 760, 1.536 s transients). While our results demonstrated that molecular coverage and dynamic range capabilities were greater in MALDI analysis, nano-DESI provided superior mass error, and all annotations for both modes had sub-ppm error. Taken together, these experiments highlight the coverage of 1676 lipids and serve as a functional guide for expected lipidome complexity within nano-DESI-MSI and MALDI-MSI. To further assess the lipidome complexity, mass splits (i.e., the difference in mass between neighboring peaks) within single pixels were collated across all pixels from each respective MSI experiment. The spatial localization of these mass splits was powerful in informing whether the observed mass splits were biological or artificial (e.g., matrix related). Mass splits down to 2.4 mDa were observed (i.e., sodium adduct ambiguity) in each experiment, and both modalities highlighted comparable degrees of lipidome complexity. Further, we highlight the persistence of certain mass splits (e.g., 8.9 mDa; double bond ambiguity) independent of ionization biases. We also evaluate the need for ultrahigh mass resolving power for mass splits ≤4.6 mDa (potassium adduct ambiguity) at m/z > 1000, which may only be resolved by advanced FTICR-MS instrumentation.

Spatially Resolved Top-Down Proteomics of Tissue Sections Based on a Microfluidic Nanodroplet Sample Preparation Platform.

Conventional proteomic approaches measure the averaged signal from mixed cell populations or bulk tissues, leading to the dilution of signals arising from subpopulations of cells that might serve as important biomarkers. Recent developments in bottom-up proteomics have enabled spatial mapping of cellular heterogeneity in tissue microenvironments. However, bottom-up proteomics cannot unambiguously define and quantify proteoforms, which are intact (i.e., functional) forms of proteins capturing genetic variations, alternatively spliced transcripts and posttranslational modifications. Herein, we described a spatially resolved top-down proteomics (TDP) platform for proteoform identification and quantitation directly from tissue sections. The spatial TDP platform consisted of a nanodroplet processing in one pot for trace samples-based sample preparation system and an laser capture microdissection-based cell isolation system. We improved the nanodroplet processing in one pot for trace samples sample preparation by adding benzonase in the extraction buffer to enhance the coverage of nucleus proteins. Using ∼200 cultured cells as test samples, this approach increased total proteoform identifications from 493 to 700; with newly identified proteoforms primarily corresponding to nuclear proteins. To demonstrate the spatial TDP platform in tissue samples, we analyzed laser capture microdissection-isolated tissue voxels from rat brain cortex and hypothalamus regions. We quantified 509 proteoforms within the union of top-down mass spectrometry-based proteoform identification and characterization and TDPortal identifications to match with features from protein mass extractor. Several proteoforms corresponding to the same gene exhibited mixed abundance profiles between two tissue regions, suggesting potential posttranslational modification-specific spatial distributions. The spatial TDP workflow has prospects for biomarker discovery at proteoform level from small tissue sections.

193 nm Ultraviolet Photodissociation for the Characterization of Singly Charged Proteoforms Generated by MALDI.

MALDI imaging allows for the near-cellular profiling of proteoforms directly from microbial, plant, and mammalian samples. Despite detecting hundreds of proteoforms, identification of unknowns with only intact mass information remains a distinct challenge, even with high mass resolving power and mass accuracy. To this end, many supplementary methods have been used to create experimental databases for accurate mass matching, including bulk or spatially resolved bottom-up and/or top-down proteomics. Herein, we describe the application of 193 nm ultraviolet photodissociation (UVPD) for fragmentation of quadrupole isolated singly charged ubiquitin (m/z 8565) by MALDI-UVPD on a UHMR HF Orbitrap. This platform permitted the high-resolution accurate mass measurement of not just terminal fragments but also large internal fragments. The outlined workflow demonstrates the feasibility of top-down analyses of isolated MALDI protein ions and the potential toward more comprehensive characterization of proteoforms in MALDI imaging applications.

Enhanced Spatial Mapping of Histone Proteoforms in Human Kidney Through MALDI-MSI by High-Field UHMR-Orbitrap Detection.

Core histones including H2A, H2B, H3, and H4 are key modulators of cellular repair, transcription, and replication within eukaryotic cells, playing vital roles in the pathogenesis of disease and cellular responses to environmental stimuli. Traditional mass spectrometry (MS)-based bottom-up and top-down proteomics allows for the comprehensive identification of proteins and of post-translational modification (PTM) harboring proteoforms. However, these methodologies have difficulties preserving near-cellular spatial distributions because they typically require laser capture microdissection (LCM) and advanced sample preparation techniques. Herein, we coupled a matrix-assisted laser desorption/ionization (MALDI) source with a Thermo Scientific Q Exactive HF Orbitrap MS upgraded with ultrahigh mass range (UHMR) boards for the first demonstration of complementary high-resolution accurate mass (HR/AM) measurements of proteoforms up to 16.5 kDa directly from tissues using this benchtop mass spectrometer. The platform achieved isotopic resolution throughout the detected mass range, providing confident assignments of proteoforms with low ppm mass error and a considerable increase in duty cycle over other Fourier transform mass analyzers. Proteoform mapping of core histones was demonstrated on sections of human kidney at near-cellular spatial resolution, with several key distributions of histone and other proteoforms noted within both healthy biopsy and a section from a renal cell carcinoma (RCC) containing nephrectomy. The use of MALDI-MS imaging (MSI) for proteoform mapping demonstrates several steps toward high-throughput accurate identification of proteoforms and provides a new tool for mapping biomolecule distributions throughout tissue sections in extended mass ranges.

Direct Infusion Metabolomics of the Photosystem and Chlorophyll Related Metabolites within a Drought Tolerant Plant Introduction of Glycine max.

Drought is the most prolific form of abiotic stress that legumes and cereal plants alike can endure, and the planting of an improper cultivar at the beginning of a season can cause unexpected losses up to fifty percent under water deficient conditions. Herein, a plant introduction (PI) of an exotic cultivar of soybean (Glycine max), PI 567731, which demonstrates a slow wilting (SW) canopy phenotype in maturity group III, was profiled under drought conditions in field trials in Missouri against a drought susceptible check cultivar, Pana. Metabolomic profiling was carried out on samples of leaves from each of these cultivars at V5 and R2 growth stages both while irrigated and while under drought stress for three weeks. PI 567731 was observed to have differential phytochemical content, and enhanced levels of chlorophyll (Chl) a/b and pheophytin (Pheo) were profiled by direct infusion electrospray Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS). Indicating drought induced changes of the photosystem and photosynthetic capabilities alongside water preservation strategies are important within the SW phenotype drought response. Subsequent multivariate analysis was able to form predictive models, encompassing the variance of growth and drought stress of the cultivar. Moreover, the existence of unique Chl-related metabolites (CRM) (m/z > 900) were confirmed through tandem mass spectrometry. The resultant coordination of fatty acids to the core of the porphyrin ring was observed and played an unknown role in the proliferation of the photosynthesis. However, the relative ratio of the most abundant CRM is undisturbed by drought stress in PI 567731, in contrast to the drought susceptible cultivar. These results provide key insights into drought related metabolic mechanisms.

Streamlined Multimodal DESI and MALDI Mass Spectrometry Imaging on a Singular Dual-Source FT-ICR Mass Spectrometer.

The study of biological specimens by mass spectrometry imaging (MSI) has had a profound influence in the various forms of spatial-omics over the past two decades including applications for the identification of clinical biomarker analysis; the metabolic fingerprinting of disease states; treatment with therapeutics; and the profiling of lipids, peptides and proteins. No singular approach is able to globally map all biomolecular classes simultaneously. This led to the development of many complementary multimodal imaging approaches to solve analytical problems: fusing multiple ionization techniques, imaging microscopy or spectroscopy, or local extractions into robust multimodal imaging methods. However, each fusion typically requires the melding of analytical information from multiple commercial platforms, and the tandem utilization of multiple commercial or third-party software platforms-even in some cases requiring computer coding. Herein, we report the use of matrix-assisted laser desorption/ionization (MALDI) in tandem with desorption electrospray ionization (DESI) imaging in the positive ion mode on a singular commercial orthogonal dual-source Fourier transform ion cyclotron resonance (FT-ICR) instrument for the complementary detection of multiple analyte classes by MSI from tissue. The DESI source was 3D printed and the commercial Bruker Daltonics software suite was used to generate mass spectrometry images in tandem with the commercial MALDI source. This approach allows for the generation of multiple modes of mass spectrometry images without the need for third-party software and a customizable platform for ambient ionization imaging. Highlighted is the streamlined workflow needed to obtain phospholipid profiles, as well as increased depth of coverage of both annotated phospholipid, cardiolipin, and ganglioside species from rat brain with both high spatial and mass resolution.

Integration of 3D-printing for a desorption electrospray ionization source for mass spectrometry.

The field of ambient ionization mass spectrometry has witnessed the development of many novel and capable methods for the analysis and imaging of surfaces, with desorption electrospray ionization being a prominent technique that has been commercialized. The adaptation of this technique to existing mass spectrometry platforms requires a laboratory-built solution manufactured with the capability of fine, stable adjustments of the electrospray emitter for liquid or solid sampling purposes. The development, fabrication, and machining require tens of hours of labor for many custom solutions. Herein described is a highly modifiable alternative approach for the fabrication of a desorption electrospray ionization source, using computer-aided design and fused deposition modeling to three-dimensionally print a source platform that utilizes standard accessories of a commercial Bruker Daltonics mass spectrometer. Three-dimensional printing allows for the inexpensive, rapid development of highly modifiable plastic parts, with the total printing time of the apparatus requiring a singular day and only a few dollars of material using a consumer grade printer. To demonstrate the utility of this printed desorption electrospray ionization source, it was fitted on an unmodified Fourier transform ion cyclotron resonance mass spectrometer for a lipid fingerprint analysis in serial sections of rat brain tissue, with the acquisition of line scans of dye-coated slides for the demonstration of serial acquisition.

MALDI Mass Spectrometry Imaging in a Primary Demyelination Model of Murine Spinal Cord.

Destruction of myelin, or demyelination, is a characteristic of traumatic spinal cord injury and pathognomonic for primary demyelinating pathologies such as multiple sclerosis (MS). The regenerative process known as remyelination, which can occur following demyelination, fails as MS progresses. Models of focal demyelination by local injection of gliotoxins have provided important biological insights into the demyelination/remyelination process. Here, injection of lysolecithin to induce spinal cord demyelination is investigated using matrix-assisted laser desorption/ionization mass spectrometry imaging. A segmentation analysis revealed changes to the lipid composition during lysolecithin-induced demyelination at the lesion site and subsequent remyelination over time. The results of this study can be utilized to identify potential myelin-repair mechanisms and in the design of therapeutic strategies to enhance myelin repair.