Compositional diversity is a fundamental property in cell populations. Single‐cell analysis promises new insight into this cellular heterogeneity on the genomic, transcriptomic, proteomic, and ...metabolomic levels. Mass spectrometry (MS) is a label‐free technique that enables the multiplexed analysis of proteins, peptides, lipids, and metabolites in individual cells. The abundances of these molecular classes are correlated with the physiological states and environmental responses of the cells. In this Minireview, we discuss recent advances in single‐cell MS techniques with an emphasis on sampling and ionization methods developed for volume‐limited samples. Strategies for sample treatment, separation methods, and data analysis require special considerations for single cells. Ongoing analytical challenges include subcellular heterogeneity, non‐normal statistical distributions of cellular properties, and the need for high‐throughput, high molecular coverage and minimal perturbation.
Singled out: Mass spectrometry (MS) has become an important tool to explore cellular heterogeneity in terms of metabolomics and proteomics. This Minireview highlights recent advances in single‐cell MS with a focus on micromanipulation, nanofabrication, and rapid gas‐phase separation techniques.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Mass spectrometric analysis of biomolecules under ambient conditions promises to enable the in vivo investigation of diverse biochemical changes in organisms with high specificity. Here we report on ...a novel combination of infrared laser ablation with electrospray ionization (LAESI) as an ambient ion source for mass spectrometry. As a result of the interactions between the ablation plume and the spray, LAESI accomplishes electrospray-like ionization. Without any sample preparation or pretreatment, this technique was capable of detecting a variety of molecular classes and size ranges (up to 66 kDa) with a detection limit of 8 and 25 fmol for verapamil and reserpine, respectively, and quantitation capabilities with a four-decade dynamic range. We demonstrated the utility of LAESI in a broad variety of applications ranging from plant biology to clinical analysis. Proteins, lipids, and metabolites were identified, and antihistamine excretion was followed via the direct analysis of bodily fluids (urine, blood, and serum). We also performed in vivo spatial profiling (on leaf, stem, and root) of metabolites in a French marigold (Tagetes patula) seedling.
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Depending on age, phase in the cell cycle, nutrition, and environmental factors, individual cells exhibit large metabolic diversity. To explore metabolic variations in cell populations, laser ...ablation electrospray ionization (LAESI) mass spectrometry (MS) was used for the in situ analysis of individual cells at atmospheric pressure. Single cell ablation was achieved by delivering mid-IR laser pulses through the etched tip of a GeO2-based glass fiber. Metabolic analysis was performed from single cells and small cell populations of Allium cepa and Narcissus pseudonarcissus bulb epidermis, as well as single eggs of Lytechinus pictus. Of the 332 peaks detected for A. cepa, 35 were assigned to metabolites with the help of accurate ion masses and tandem MS. The metabolic profiles from single cells of the two plant species included a large variety of oligosaccharides including possibly fructans in A. cepa, and alkaloids, e.g., lycorine in N. pseudonarcissus. Analysis of adjacent individual cells with a difference in pigmentation showed that, in addition to essential metabolites found in both variants, the pigmented cells contained anthocyanidins, other flavonoids, and their glucosides. Analysis of single epidermal cells from different scale leaves in an A. cepa bulb showed metabolic differences corresponding to their age. Our results indicate the feasibility of using LAESI-MS for the in situ analysis of metabolites in single cells with potential applications in studying cell differentiation, changes due to disease states, and response to xenobiotics.
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► Ambient MS emerges to explore biological systems under native conditions to specimens. ► We use the ambient MS framework to gauge trends in in vivo and in situ applications. ► We probe the ...organism–organ-tissue–cell–organelle realm on a biochemical level. ► We unveil specimen chemistry in time, and in two and three spatial dimensions. ► We identify analytical challenges and developmental opportunities for biochemistry.
Recent technical innovations in mass spectrometry (MS) have extended the application of this powerful technique to direct chemical analysis at atmospheric pressure. These innovations have created an opportunity to appreciate the chemistry of biological systems in their native state, so tissues and single cells of plant, animal, or human origin can be interrogated in situ and in vivo.
Ambient MS also allows label-free detection of compounds and gives unique insights into temporal changes and tissue architecture in two and three dimensions. Compounds studied range from natural products (e.g., neurotransmitters, metabolites, organic acids, polyamines, sugars, lipids, and peptides) to xenobiotics (e.g., pharmaceuticals), dyes, polymers, explosives, and toxins.
This critical review covers analytical trends in ambient MS. Our discussions primarily touch on the mechanisms of sampling and the bioanalytical implications for in situ and in vivo experiments. We pay special attention to lateral imaging, depth profiling, and three-dimensional-MS imaging, all while working under atmospheric conditions. Our closing remarks highlight some of the present analytical challenges and developmental opportunities in this field.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Metabolism in microbial colonies responds to competing species, rapidly evolving genetic makeup, and sometimes dramatic environmental changes. Conventional characterization of the existing and ...emerging microbial strains and their interactions with antimicrobial agents, e.g., the Kirby–Bauer susceptibility test, relies on time consuming methods with limited ability to discern the molecular mechanism and the minimum inhibitory concentration. Assessing the metabolic adaptation of microbial colonies requires their non‐targeted molecular imaging in a native environment. Laser ablation electrospray ionization (LAESI) is an ambient ionization technique that in combination with mass spectrometry (MS) enables the analysis and imaging of numerous metabolites and lipids. In this contribution, we report on the application of LAESI‐MS imaging to gain deeper molecular insight into microbe–antibiotic interactions, and enhance the quantitative nature of antibiotic susceptibility testing while significantly reducing the required incubation time.
Visualizing the interaction zone: Laser ablation electrospray ionization (LAESI) in combination with mass spectrometry (MS) was applied to gain deeper molecular insight into microbe–antibiotic interactions. LAESI‐MS imaging enhances the quantitative nature of antibiotic susceptibility testing while significantly reducing the required incubation time.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Atmospheric pressure imaging mass spectrometry is a rapidly expanding field that offers advantages in the ability to study biological systems in their native condition, simplified sample preparation, ...and high-throughput experiments. In laser ablation electrospray ionization (LAESI), the native water molecules in biological tissues facilitate sampling by a focused mid-infrared laser beam. The ionization of the ablated material is accomplished by electrospray postionization. In this work, we demonstrate that the imaging variant of LAESI simultaneously provides lateral distributions for small metabolites and lipids directly in rat brain sections. To cope with the fragile nature and potential dehydration of the brain tissue due to drying in the ambient environment as well as to minimize analyte redistribution, a Peltier cooling stage is integrated into the LAESI imaging system. We demonstrate the utility of high-resolution (m/Δm > 6000) time-of-flight mass spectrometry with LAESI to deconvolute spatial distributions of different chemical species with identical nominal mass. To help with the evaluation of the massive data sets, Pearson colocalization maps are calculated for selected small metabolites and lipids. We show that this approach reveals biologically meaningful correlations between these two classes of biomolecules.
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Microbial colonization of medical devices is a widespread problem that tests the limits of conventional analytical methods. Successful analytical endeavors require collaboration between clinicians, ...microbiologists, biomedical engineers, and analytical chemists.
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Three-dimensional (3D) imaging of molecular distributions offers insight into the correlation between biochemical processes and the spatial organization of a biological tissue. Simultaneous ...identification of diverse molecules is a virtue of mass spectrometry (MS) that in combination with ambient ion sources enables the atmospheric pressure investigation of biomolecular distributions and processes. Here, we report on the development of an MS-based technique that allows 3D chemical imaging of tissues under ambient conditions without sample preparation. The method utilizes laser ablation electrospray ionization (LAESI) for direct molecular imaging with lateral and depth resolutions of ∼300 μm and 30−40 μm, respectively. We demonstrate the feasibility of LAESI 3D imaging MS of metabolites in the leaf tissues of Peace lily (Spathiphyllum lynise) and the variegated Zebra plant (Aphelandra squarrosa). Extensive tandem MS studies help with the structure identification of the metabolites. The 3D distributions are found to exhibit tissue-specific metabolite accumulation patterns that correlate with the biochemical roles of these chemical species in plant defense and photosynthesis. Spatial correlation coefficients between the intensity distributions of different ions help to identify colocalization of metabolites and potentially uncover connections between metabolic pathways.
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Ambient ionization methods for mass spectrometry have enabled the in situ and in vivo analysis of biological tissues and cells. When an etched optical fiber is used to deliver laser energy to a ...sample in laser ablation electrospray ionization (LAESI) mass spectrometry, the analysis of large single cells becomes possible. However, because in this arrangement the ablation plume expands in three dimensions, only a small portion of it is ionized by the electrospray. Here we show that sample ablation within a capillary helps to confine the radial expansion of the plume. Plume collimation, due to the altered expansion dynamics, leads to greater interaction with the electrospray plume resulting in increased ionization efficiency, reduced limit of detection (by a factor of ∼13, reaching 600 amol for verapamil), and extended dynamic range (6 orders of magnitude) compared to conventional LAESI. This enhanced sensitivity enables the analysis of a range of metabolites from small cell populations and single cells in the ambient environment. This technique has the potential to be integrated with flow cytometry for high-throughput metabolite analysis of sorted cells.
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The utility of atmospheric pressure infrared MALDI mass spectrometry (AP IR-MALDI) was assessed for plant metabolomics studies. Tissue sections from plant organs, including flowers, ovaries, ...aggregate fruits, fruits, leaves, tubers, bulbs, and seeds were studied in both positive and negative ion modes. For leaves, single laser pulses sampled the cuticle and upper epidermal cells, whereas multiple pulses were demonstrated to ablate some mesophyll layers. Tandem mass spectra were obtained with collision-activated dissociation to aid with the identification of some observed ions. In the positive mode, most ions were produced as potassium, proton, or sometimes sodium ion adducts, whereas proton loss was dominant in the negative ion mode. Over 50 small metabolites and various lipids were detected in the spectra including, for example, 7 of the 10 intermediates in the citric acid cycle. Key components of the glycolysis pathway occurring in the plant cytosol were found along with intermediates of phospholipid biosynthesis and reactants or products of amino acid, nucleotide, oligosaccharide, and flavonoid biosynthesis. AP IR-MALDI mass spectrometry was used to follow the fluid transport driven by transpiration and image the spatial distributions of several metabolites in a white lily (Lilium candidum) flower petal.
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