The authors acknowledge financial support by the following institutions; the National Science Foundation (Grant 0719232), the Department of Energy (Grant DEFG02-01ER15129), the W. M. Keck Foundation (Grant 041904), and the George Washington University Research Enhancement Fund. The authors are grateful for the GeO2-based optical fibers generously supplied by Infrared Fiber Systems (Silver Spring, MD) as well as for the initial discussion on the protocol regarding fiber etching by Mark E. Reeves and Joan A. Hoffmann of the George Washington University. The authors would also like to thank Jessica A. Stolee for her help during the videotaping of the protocol.

1. Optical Components
<ol>
<li>	Laser ablation for laser ablation electrospray ionization (LAESI) mass spectrometry (MS) is produced by a 5 ns laser pulse at 2.94 &mu;m wavelength. A tunable optical parametric oscillator driven by a Nd:YAG laser (Opolette 100, Opotek Inc., Carlsbad, CA) is used for ablation in this study. The repetition rate is selected between 5 and 100 Hz. This is a class IV laser that upon direct exposure may cause severe permanent damage to the eye or skin. The diffuse reflection of the laser beam can also be hazardous to the skin or eye. Wear an appropriate laser goggle for protection. </li>
<li>	The optical fiber made of germanium dioxide-based glass was obtained from Infrared Fiber Systems, Inc. (Silver Spring, MD). If there are Hytrel and polyimide coatings on the fiber follow the directions below to remove them from the ends. Heat 1-methyl-2-pyrrolidinone to 130-150 &deg;C in a fume hood. Dip the fiber ends you want to strip into this liquid for approximately 1 min or until the coating starts to soften and peel off. Dip the softened coating into methanol or isopropanol for a minute, and wipe off any remaining coating with a lint-free napkin. After removing the coating, cleave both ends of the fiber with a sapphire blade (KITCO Fiber Optics, Virginia Beach, VA) by scoring and gently snapping them.</li>
<li>	To achieve the desired sharpness, chemically etch one end of the fiber tip by 1% (v/v) nitric acid (reagent grade) solution. Vertically immerse the selected end of the fiber into the acid solution to a depth of 300 to 500 &mu;m after the initial contact. After approximately 15 minutes of etching, the tip will spontaneously detach from the acid surface. This results in a sharpened fiber tip with ~15 &mu;m radius of curvature. Rinse with deionized water to remove any acid residue. </li>
<li>	Mount the etched end of the fiber on a micromanipulator (MN-151, Narishige, Tokyo, Japan) and bring it to close proximity (~20 &mu;m) of the cell surface for efficient energy deposition.</li>
<li>	Hold the non-etched end of the optical fiber by a bare fiber chuck (BFC300, Siskiyou Corporation, Grants Pass, OR). For optical alignment, the fiber chuck is mounted on a five-axis translator (BFT-5, Siskiyou Corporation, Grants Pass, OR). The laser energy (0.5 to 1 mJ) is coupled into the fiber by focusing the beam by a plano-convex calcium fluoride or antireflection coated ZnSe lens (Infrared Optical Products, Farmingdale, NY) onto its non-etched end. Protected gold mirrors (PF10-03-M01, Thorlabs, Newton, NJ) are used to steer the mid-IR laser beam onto the focusing lens.</li>
</ol>
2. Electrospray Setup
<ol>
<li>	The electrospray system can be constructed using the following components: metal union with conductive perfluoroelastomer ferrule, fittings, tubing sleeve, needle port (e.g., U-435, M215,F-331Nx, F-242x, 9013, respectively IDEX Health &amp; Sciences, Oak Harbor, WA) as well as fused silica tubing (e.g., CT360-100-50-5, New Objective Inc., Woburn, MA). The recommended emitter is made of stainless steel and has a tapered tip with a 50 &mu;m i.d. (e.g., MT320-50-5-5, New Objective Inc., Woburn, MA). </li>
<li>	Prepare 50% methanol solution with 0.1 % acetic acid (v/v) for the electrospray. The solution is pumped at a rate of 200 to 300 nl/min by a syringe pump (Harvard Apparatus, Holliston, MA) using, e.g., a 500 &mu;l syringe (81222, Hamilton Company, Reno, NV).</li>
<li>	Apply high voltage between 2,800 and 3,000 V to the metal union by a regulated power supply (PS350, Stanford Research Systems Inc, Sunnyvale, CA) to generate a steady electrospray. Make sure all electric connections are secure and shielded with the shielding properly grounded. Direct contact with exposed high voltage can result in electric shock. </li>
</ol>
3. Sample Handling
<ol>
<li>	Obtain the live tissue or cell sample from an appropriate source and keep them as recommended before the analysis. The Allium cepa bulbs for this demonstration were purchased from a local store. Cut a bulb longitudinally by a surgical scalpel. A few centimeter segment of a scale was sectioned into a strip. An intact monolayer of the inner epidermal tissue is peeled off. </li>
<li>	The wet surface of the epidermis is used to mount the tissue on a pre-cleaned glass microscope slide. Hold the slide by a general purpose plate holder (FP01, Thorlabs Inc., Newton, NJ) mounted on a three-axis translation stage for positioning. Acquire the mass spectra immediately after mounting the sample to prevent cell degradation.</li>
</ol>
4. Visualization System
<ol>
<li>	Two long distance microscopes are used to select the cells for analysis and to maintain the distance between the sharpened fiber tip and the cell surface. The latter is mounted under 15&deg;-30&deg; angle measured from the surface. This system consist of a long distance video microscope (7x precision zoom optic, Edmund Optics, Barrington, NJ) with a 5x infinity corrected objective lens (M Plan Apo 5x, Mitutoyo Co., Kanagawa, Japan) equipped with a CCD camera (Marlin F131, Allied Vision Technologies, Stadtroda, Germany) for monitoring and image capture.</li>
<li>	The second video microscope is mounted at a right angle to the sample surface to visualize the cells from the top. This system provides visual feedback to align the fiber tip over the cell selected for ablation and analysis. It consists of a 7x precision zoom optic (Edmund Optics, Barrington, NJ), fitted with a 10x infinity corrected long working distance objective lens (M Plan Apo 10x, Mitutoyo Co., Kanagawa, Japan) and a CCD camera.</li>
</ol>
5. Acquisition of Mass Spectra
<ol>
<li>	Turn on the laser, the electrospray and the mass spectrometer, e.g., a quadrupole time-of-flight system (Q-TOF Premier, Waters, Milford, MA). Optimize the geometry of the LAESI ion source by adjusting the position of the ablation spot and electrospray emitter relative to each other and the orifice of the mass spectrometer to achieve maximum ion intensities.</li> 
<li>	Select the cell of interest for analysis by using the top-view visualization system. After selection, move the sample stage to bring the selected cell directly below the sharpened tip of the optical fiber. Using the side-view microscope readjust the tip-to-cell surface distance to ~20 &mu;m. </li>
<li>	Fire laser pulses at the selected cell through the etched fiber tip. This result in the perforation of the cell wall and the ejection of the cytoplasm in the form of particulate matter. After postionization by the electrospray, the produced ions are collected by the mass spectrometer and mass spectra are recorded. </li>
<li>	After data acquisition, put the laser, the electrospray and the mass spectrometer in standby mode or turn them off. Keep the ablated A. cepa tissue sample for optional observation by light microscopy. </li>
</ol>
6. Representative Results
Successful ablation of a single cell results in the bursting of the cell wall and the collection of a LAESI mass spectrum. An unsuccessful experiment usually results in no ablation and/or no mass spectrum. For demonstration purposes, we present the results from the LAESI-MS analysis of a single embedded epidermal cell of an A. cepa bulb. Figure 2a shows a representative optical microscopy image after the sampling of a cell. The cell wall is perforated and the extent of perforation is limited by the boundaries with the neighbouring cells. The adjacent cells seem to be intact. Figure 2b shows a representative LAESI mass spectrum produced from the cell. A wide variety of ions are detected from the A. cepa cell and based on their accurate masses, isotope distribution patterns, and, in some cases, tandem mass spectra, they are tentatively assigned to endogenous metabolites. The ions detected from a single cell were similar to the ones observed in the analysis of the same tissue by conventional LAESI-MS of multiple cells.13
<img src="/files/ftp_upload/2144/2144fig1.jpg" alt="Figure 1" /> 
Figure 1. Schematic of single cell analysis by LAESI-MS. A mid-IR laser pulse is coupled into an optical fiber and delivered to the sample placed on glass slide. The sharpened fiber tip focuses the light into the target cell and the deposited energy bursts the cell. The produced ablation plume (red dots) is merged with an electrospray plume (black dots) the droplets coalesce (green dots) resulting in sample specific ion production. These ions are analyzed and detected by the mass spectrometer (MS). Two long distance video microscopes, Microscope 1 and Microscope 2, are utilized to monitor distance between the fiber tip and the cell surface and to select a cell for analysis, respectively. 
<img src="/files/ftp_upload/2144/2144fig2a.jpg" alt="Figure 2a" /> 
Figure 2a. Ablation mark on a single epidermal cell of an A. cepa bulb. The adjacent cells do not exhibit visible damage and subsequently can be analyzed separately.
<img src="/files/ftp_upload/2144/2144fig2b.jpg" alt="Figure 2b" /> 
Figure 2b. Mass spectrum corresponding to the ablation of a single A. cepa epidermal cell shows the presence of primary and secondary metabolites.

<ol>
	<li>Cooks, R. G., Ouyang, Z., Takats, Z., Wiseman, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ambient+Mass+Spectrometry.">Ambient Mass Spectrometry.</a> Science. 311, 1566-1570 (2006).</li><li>Chen, Z. Y., Vertes, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+plume+expansion+in+atmospheric+pressure+midinfrared+laser+ablation+of+water-rich+targets.">Early plume expansion in atmospheric pressure midinfrared laser ablation of water-rich targets.</a> Physical Review E. 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Analytical Chemistry. 79, 523-532 (2006).</li><li>Li, Y., Shrestha, B., Vertes, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atmospheric+Pressure+Infrared+MALDI+Imaging+Mass+Spectrometry+for+Plant+Metabolomics.">Atmospheric Pressure Infrared MALDI Imaging Mass Spectrometry for Plant Metabolomics.</a> Analytical Chemistry. 80, 407-420 (2007).</li><li>Nemes, P., Vertes, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+Ablation+Electrospray+Ionization+for+Atmospheric+Pressure+in+Vivo,+and+Imaging+Mass+Spectrometry.">Laser Ablation Electrospray Ionization for Atmospheric Pressure in Vivo, and Imaging Mass Spectrometry.</a> Analytical Chemistry. 79, 8098-8106 (2007).</li><li>Shrestha, B., Li, Y., Vertes, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+analysis+of+pharmaceuticals+and+excreted+xenobiotic+and+endogenous+metabolites+with+atmospheric+pressure+infrared+MALDI+mass+spectrometry.">Rapid analysis of pharmaceuticals and excreted xenobiotic and endogenous metabolites with atmospheric pressure infrared MALDI mass spectrometry.</a> Metabolomics. 4, 297-311 (2008).</li><li>Nemes, P., Barton, A. A., Li, Y., Vertes, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ambient+Molecular+Imaging+and+Depth+Profiling+of+Live+Tissue+by+Infrared+Laser+Ablation+Electrospray+Ionization+Mass+Spectrometry.">Ambient Molecular Imaging and Depth Profiling of Live Tissue by Infrared Laser Ablation Electrospray Ionization Mass Spectrometry.</a> Analytical Chemistry. 80, 4575-4582 (2008).</li><li>Nemes, P., Barton, A. 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No conflicts of interest declared.

The water content of the cell cytoplasm and the tensile strength of the cell wall or membrane are critical factors governing the ablation of single cells during LAESI-MS analysis. The laser fluence has to be adjusted to reach the ablation thresholds of the different cell types. The delivered laser fluence depends on the energy of the laser pulse and on the distance between the fiber tip and the cell surface. Minimizing the perturbation of neighboring cells during the single cell analysis is critical. Keeping the laser energy at the ablation threshold limits the effect of analysis on the adjacent cells.
One of the potential applications is using single cells as the natural volumetric pixels (voxels) for chemical imaging by MS. Compared to collecting data on an artificial often rectangular grid, chemical imaging of a tissue one cell at a time is more likely to retain the spatial organization of biochemical interactions between cells.

<table><tbody><tr><td>Acetic acid</td><td>Fluka</td><td>45727</td><td></td></tr><tr><td>Methanol</td><td>Fluka</td><td>65548</td><td></td></tr><tr><td>1-methyl-2-pyrrolidinon</td><td>Sigma-Aldrich</td><td>M79603</td><td></td></tr><tr><td>Water</td><td>Fluka</td><td>39259</td><td></td></tr></tbody></table>

direct analysis of single cells by mass spectrometry at atmospheric pressure

Analysis of biochemicals in single cells is important for understanding cell metabolism, cell cycle, adaptation, disease states, etc. Even the same cell types exhibit heterogeneous biochemical makeup depending on their physiological conditions and interactions with the environment. Conventional methods of mass spectrometry (MS) used for the analysis of biomolecules in single cells rely on extensive sample preparation. Removing the cells from their natural environment and extensive sample processing could lead to changes in the cellular composition. Ambient ionization methods enable the analysis of samples in their native environment and without extensive sample preparation.1 The techniques based on the mid infrared (mid-IR) laser ablation of biological materials at 2.94 &mu;m wavelength utilize the sudden excitation of water that results in phase explosion.2 Ambient ionization techniques based on mid-IR laser radiation, such as laser ablation electrospray ionization (LAESI) and atmospheric pressure infrared matrix-assisted laser desorption ionization (AP IR-MALDI), have successfully demonstrated the ability to directly analyze water-rich tissues and biofluids at atmospheric pressure.3-11 In LAESI the mid-IR laser ablation plume that mostly consists of neutral particulate matter from the sample coalesces with highly charged electrospray droplets to produce ions. Recently, mid-IR ablation of single cells was performed by delivering the mid-IR radiation through an etched fiber. The plume generated from this ablation was postionized by an electrospray enabling the analysis of diverse metabolites in single cells by LAESI-MS.12 This article describes the detailed protocol for single cell analysis using LAESI-MS. The presented video demonstrates the analysis of a single epidermal cell from the skin of an Allium cepa bulb. The schematic of the system is shown in Figure 1. A representative example of single cell ablation and a LAESI mass spectrum from the cell are provided in Figure 2.

Watch this Scientific Journal Video about Direct Analysis of Single Cells by Mass Spectrometry at Atmospheric Pressure at JoVE.com

Direct Analysis of Single Cells by Mass Spectrometry at Atmospheric Pressure

Single cell analysis is performed by mass spectrometry on plant and animal cells at atmospheric pressure by using a sharpened optical fiber to sample the cells for laser ablation electrospray ionization (LAESI) mass spectrometry.

Analysis of biochemicals in single cells is important for understanding cell metabolism, cell cycle, adaptation, disease states, etc. Even the same cell ...

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15.9K Views.  George Washington University. Single cell analysis is performed by mass spectrometry on plant and animal cells at atmospheric pressure by using a sharpened optical fiber to sample the cells for laser ablation electrospray ionization (LAESI) mass spectrometry.

Video: Direct Analysis of Single Cells by Mass Spectrometry at Atmospheric Pressure

Rapid High-throughput Species Identification of Botanical Material Using Direct Analysis in Real Time High Resolution Mass Spectrometry

We demonstrate that direct analysis in real time-high resolution mass spectrometry can be used to produce mass spectral profiles of botanical material, and that these chemical fingerprints can be used for plant species identification. The mass spectral data can be acquired rapidly and in a high throughput manner without the need for sample extraction, derivatization or pH adjustment steps. The use of this technique bypasses challenges presented by more conventional techniques including lengthy chromatography analysis times and resource intensive methods. The high throughput capabilities of the direct analysis in real time-high resolution mass spectrometry protocol, coupled with multivariate statistical analysis processing of the data, provide not only class characterization of plants, but also yield species and varietal information. Here, the technique is demonstrated with two psychoactive plant products, Mitragyna speciosa (Kratom) and Datura (Jimsonweed), which were subjected to direct analysis in real time-high resolution mass spectrometry followed by statistical analysis processing of the mass spectral data. The application of these tools in tandem enabled the plant materials to be rapidly identified at the level of variety and species.

A method for species identification of botanical material by direct analysis in real time-high resolution mass spectrometry and multivariate statistical analysis is presented.

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Microprobe Capillary Electrophoresis Mass Spectrometry for Single-cell Metabolomics in Live Frog (Xenopus laevis) Embryos

The quantification of small molecules in single cells raises new potentials for better understanding the basic processes that underlie embryonic development. To enable single-cell investigations directly in live embryos, new analytical approaches are needed, particularly those that are sensitive, selective, quantitative, robust, and scalable to different cell sizes. Here, we present a protocol that enables the in situ analysis of metabolism in single cells in freely developing embryos of the South African clawed frog (Xenopus laevis), a powerful model in cell and developmental biology. This approach uses a capillary microprobe to aspirate a defined portion from single identified cells in the embryo, leaving neighboring cells intact for subsequent analysis. The collected cell content is analyzed by a microscale capillary electrophoresis electrospray ionization (CE-ESI) interface coupled to a high-resolution tandem mass spectrometer. This approach is scalable to various cell sizes and compatible with the complex three-dimensional structure of the developing embryo. As an example, we demonstrate that microprobe single-cell CE-ESI-MS enables the elucidation of metabolic cell heterogeneity that unfolds as a progenitor cell gives rise to descendants during development of the embryo. Besides cell and developmental biology, the single-cell analysis protocols described here are amenable to other cell sizes, cell types, or animal models.

Microprobe Capillary Electrophoresis Mass Spectrometry for Single-cell Metabolomics in Live Frog Xenopus laevis Embryos

We describe steps that enable fast in situ sampling of a small portion of an individual cell with high precision and minimal invasion using capillary-based micro-sampling, to facilitate chemical characterization of a snapshot of metabolic activity in live embryos using a custom-built single cell capillary electrophoresis and mass spectrometry platform.

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An Integrated Raman Spectroscopy and Mass Spectrometry Platform to Study Single-Cell Drug Uptake, Metabolism, and Effects

Cells are known to be inherently heterogeneous in their responses to drugs. Therefore, it is essential that single-cell heterogeneity is accounted for in drug discovery studies. This can be achieved by accurately measuring the plethora of cellular interactions between a cell and drug at the single-cell level (i.e., drug uptake, metabolism, and effect). This paper describes a single-cell Raman spectroscopy and mass spectrometry (MS) platform to monitor metabolic changes of cells in response to drugs. Using this platform, metabolic changes in response to the drug can be measured by Raman spectroscopy, while the drug and its metabolite can be quantified using mass spectrometry in the same cell. The results suggest that it is possible to access information about drug uptake, metabolism, and response at a single-cell level.

This protocol presents an integrated Raman spectroscopy-mass spectrometry (MS) platform that is capable of achieving single-cell resolution. Raman spectroscopy can be used to study cellular response to drugs, while MS can be used for targeted and quantitative analysis of drug uptake and metabolism.

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Integrated Cell Manipulation Platform Coupled with the Single-probe for Mass Spectrometry Analysis of Drugs and Metabolites in Single Suspension Cells

Single cell mass spectrometry (SCMS) enables sensitive detection and accurate analysis of broad ranges of cellular species on the individual-cell level. The single-probe, a microscale sampling and ionization device, can be coupled with a mass spectrometer for on-line, rapid SCMS analysis of cellular constituents under ambient conditions. Previously, the single-probe SCMS technique was primarily used to measure cells immobilized onto a substrate, limiting the types of cells for studies. In the current study, the single-probe SCMS technology has been integrated with a cell manipulation system, typically used for in vitro fertilization. This integrated cell manipulation and analysis platform uses a cell-selection probe to capture identified individual floating cells and transfer the cells to the single-probe tip for microscale lysis, followed by immediate mass spectrometry analysis. This capture and transfer process removes the cells from the surrounding solution prior to analysis, minimizing the introduction of matrix molecules in the mass spectrometry analysis. This integrated setup is capable of SCMS analysis of targeted patient-isolated cells present in body fluids samples (e.g., urine, blood, saliva, etc.), allowing for potential applications of SCMS analysis to human medicine and disease biology.

An integrated cell manipulation platform is developed for use in conjunction with a single-probe mass spectrometry setup for the on-line analysis of individual suspension cells under ambient conditions.

Single cell mass spectrometry (SCMS) enables sensitive detection and accurate analysis of broad ranges of cellular species on the individual-cell level. ...

Analyzing Large Protein Complexes by Structural Mass Spectrometry

Living cells control and regulate their biological processes through the coordinated action of a large number of proteins that assemble themselves into an array of dynamic, multi-protein complexes1. To gain a mechanistic understanding of the various cellular processes, it is crucial to determine the structure of such protein complexes, and reveal how their structural organization dictates their function. Many aspects of multi-protein complexes are, however, difficult to characterize, due to their heterogeneous nature, asymmetric structure, and dynamics. Therefore, new approaches are required for the study of the tertiary levels of protein organization.
One of the emerging structural biology tools for analyzing macromolecular complexes is mass spectrometry (MS)2-5. This method yields information on the complex protein composition, subunit stoichiometry, and structural topology. The power of MS derives from its high sensitivity and, as a consequence, low sample requirement, which enables examination of protein complexes expressed at endogenous levels. Another advantage is the speed of analysis, which allows monitoring of reactions in real time. Moreover, the technique can simultaneously measure the characteristics of separate populations co-existing in a mixture. 
Here, we describe a detailed protocol for the application of structural MS to the analysis of large protein assemblies. The procedure begins with the preparation of gold-coated capillaries for nanoflow electrospray ionization (nESI). It then continues with sample preparation, emphasizing the buffer conditions which should be compatible with nESI on the one hand, and enable to maintain complexes intact on the other. We then explain, step-by-step, how to optimize the experimental conditions for high mass measurements and acquire MS and tandem MS spectra. Finally, we chart the data processing and analyses that follow. Rather than attempting to characterize every aspect of protein assemblies, this protocol introduces basic MS procedures, enabling the performance of MS and MS/MS experiments on non-covalent complexes. Overall, our goal is to provide researchers unacquainted with the field of structural MS, with knowledge of the principal experimental tools.

Mass spectrometry has proven to be a valuable tool for analyzing large protein complexes. This method enables insights into the composition, stoichiometry and overall architecture of multi-subunit assemblies. Here, we describe, step-by-step, how to perform a structural mass spectrometry analysis, and characterize macromolecular structures.

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Atmospheric-pressure Molecular Imaging of Biological Tissues and Biofilms by LAESI Mass Spectrometry

Ambient ionization methods in mass spectrometry allow analytical investigations to be performed directly on a tissue or biofilm under native-like experimental conditions. Laser ablation electrospray ionization (LAESI) is one such development and is particularly well-suited for the investigation of water-containing specimens. LAESI utilizes a mid-infrared laser beam (2.94 &mu;m wavelength) to excite the water molecules of the sample. When the ablation fluence threshold is exceeded, the sample material is expelled in the form of particulate matter and these projectiles travel to tens of millimeters above the sample surface. In LAESI, this ablation plume is intercepted by highly charged droplets to capture a fraction of the ejected sample material and convert its chemical constituents into gas-phase ions. A mass spectrometer equipped with an atmospheric-pressure ion source interface is employed to analyze and record the composition of the released ions originating from the probed area (pixel) of the sample. A systematic interrogation over an array of pixels opens a way for molecular imaging in the microprobe analysis mode. A unique aspect of LAESI mass spectrometric imaging is depth profiling that, in combination with lateral imaging, enables three-dimensional (3D) molecular imaging. With current lateral and depth resolutions of ~100 &mu;m and ~40 &mu;m, respectively, LAESI mass spectrometric imaging helps to explore the molecular structure of biological tissues. Herein, we review the major elements of a LAESI system and provide guidelines for a successful imaging experiment.

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Mass spectrometry imaging (MSI) and in-situ single cell mass spectrometry (SCMS) analysis under ambient conditions are two emerging fields with great ...

Quantification of Site-specific Protein Lysine Acetylation and Succinylation Stoichiometry Using Data-independent Acquisition Mass Spectrometry

Post-translational modification (PTM) of protein lysine residues by N&#400;-acylation induces structural changes that can dynamically regulate protein functions, for example, by changing enzymatic activity or by mediating interactions. Precise quantification of site-specific protein acylation occupancy, or stoichiometry, is essential for understanding the functional consequences of both global low-level stoichiometry and individual high-level acylation stoichiometry of specific lysine residues. Other groups have reported measurement of lysine acetylation stoichiometry by comparing the ratio of peptide precursor isotopes from endogenous, natural abundance acylation and exogenous, heavy isotope-labeled acylation introduced after quantitative chemical acetylation of proteins using stable isotope-labeled acetic anhydride. This protocol describes an optimized approach featuring several improvements, including: (1) increased chemical acylation efficiency, (2) the ability to measure protein succinylation in addition to acetylation, and (3) improved quantitative accuracy due to reduced interferences using fragment ion quantification from data-independent acquisitions (DIA) instead of precursor ion signal from data-dependent acquisition (DDA). The use of extracted peak areas from fragment ions for quantification also uniquely enables differentiation of site-level acylation stoichiometry from proteolytic peptides containing more than one lysine residue, which is not possible using precursor ion signals for quantification. Data visualization in Skyline, an open source quantitative proteomics environment, allows for convenient data inspection and review. Together, this workflow offers unbiased, precise, and accurate quantification of site-specific lysine acetylation and succinylation occupancy of an entire proteome, which may reveal and prioritize biologically relevant acylation sites.

Here, we present unbiased quantification of site-specific protein acetylation and/or succinylation occupancy (stoichiometry) of an entire proteome through a ratiometric analysis of endogenous modifications to modifications introduced after quantitative chemical acylation using stable isotope-labeled anhydrides. In combination with sensitive data-independent acquisition mass spectrometry, accurate site occupancy measurements are obtained.

Post-translational modification (PTM) of protein lysine residues by N&#400;-acylation induces structural changes that can dynamically regulate protein ...

Single-Cell Proteomics Preparation for Mass Spectrometry Analysis Using Freeze-Heat Lysis and an Isobaric Carrier

Single-cell proteomics analysis requires sensitive, quantitatively accurate, widely accessible, and robust methods. To meet these requirements, the Single-Cell ProtEomics (SCoPE2) protocol was developed as a second-generation method for quantifying hundreds to thousands of proteins from limited samples, down to the level of a single cell. Experiments using this method have achieved quantifying over 3,000 proteins across 1,500 single mammalian cells (500-1,000 proteins per cell) in 10 days of mass spectrometer instrument time. SCoPE2 leverages a freeze-heat cycle for cell lysis, obviating the need for clean-up of single cells and consequently reducing sample losses, while expediting sample preparation and simplifying its automation. Additionally, the method uses an isobaric carrier, which aids protein identification and reduces sample losses.
This video protocol provides detailed guidance to enable the adoption of automated single-cell protein analysis using only equipment and reagents that are widely accessible. We demonstrate critical steps in the procedure of preparing single cells for proteomic analysis, from harvesting up to injection to liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Additionally, viewers are guided through the principles of experimental design with the isobaric carrier, quality control for both isobaric carrier and single-cell preparations, and representative results with a discussion of limitations of the approach.

In this protocol, we describe how to prepare mammalian cells for single-cell proteomics analysis via mass spectrometry using commercially available reagents and equipment, with options for both manual and automatic pipetting.

Single-cell proteomics analysis requires sensitive, quantitatively accurate, widely accessible, and robust methods. To meet these requirements, the ...

Introduction to Mass Spectrometry

Source: Laboratory of Dr. Khuloud Al-Jamal - King&#39;s College London

Mass spectrometry is an analytical chemistry technique that enables the identification of unknown compounds within a sample, the quantification of known materials, the determination of the structure, and chemical properties of different molecules.
A mass spectrometer is composed of an ionization source, an analyzer,&#160;and a detector. The process involves the ionization of chemical compounds to generate ions. When using inductively coupled plasma (ICP), samples containing elements of interest are introduced into argon plasma as aerosol droplets. The plasma dries the aerosol, dissociates the molecules, and then removes an electron from the components to be detected by the mass spectrometer. Other ionization methods such as electrospray ionization (ESI) and matrix assisted laser desorption ionization (MALDI) are used to analyze biological samples. Following the ionization procedure, ions are separated in the mass spectrometer according to their mass-to-charge ratio (m/z), and the relative abundance of each ion type is measured. Finally, the detector commonly consists in an electron multiplier where the collision of ions with a charged anode leads to a cascade of increasing number of electrons, which can be detected by an electrical circuit connected to a computer.
In this video, the procedure of ICP-MS analysis will be described by the detection of 56Fe as an example.