Name: leaf spray mass spectrometry: a rapid ambient ionization technique to directly assess metabolites from plant tissues
Uploaded: 2018-06-21T15:00:00
Description: Watch this Scientific Journal Video about Leaf Spray Mass Spectrometry: A Rapid Ambient Ionization Technique to Directly Assess Metabolites from Plant Tissues at JoVE.com

This work was funded by the NSF Plant Genome Research Program grant IOS-1238812 and the Postdoctoral Fellowship in Biology IOS-1400818. The work was also funded by a Monsanto Graduate Student Fellowship to Katherine A. Sammons. The Fulbright African Researcher Scholars Program (2017-2018) is thanked for funding awarded to Nokwanda P. Makunga. We greatly appreciate the donation of a nanospray source from Jessica Prenni and the Proteomics and Metabolomics facility at Colorado State University.

1. Modifications to Nanospray Source for Leaf Spray MS
<ol>
	<li>Use a modified nanospray source for leaf spray MS. As no fluidic components are necessary for leaf spray MS, modify the source by removing the LC probe from the source.</li>
	<li>Assemble the leaf spray MS wire that will apply the voltage to the plant tissue with the appropriate pin for plugging into the source. Solder the pin to one end of an insulated wire; solder a clamp to the opposite end of the wire. 
	NOTE: The clamp (alligator clip type) may ...

<ol>
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G., Ouyang, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leaf+spray:+direct+chemical+analysis+of+plant+material+and+living+plants+by+mass+spectrometry.">Leaf spray: direct chemical analysis of plant material and living plants by mass spectrometry.</a> Analytical Chemistry. 83 (20), 7608-7613 (2011).</li><li>Chan, S. L. -. F., Wong, M. Y. -. M., Tang, H. -. W., Che, C. -. M., Ng, K. -. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-spray+ionization+mass+spectrometry+for+raw+herb+analysis.">Tissue-spray ionization mass spectrometry for raw herb analysis.</a> Rapid Communications in Mass Spectrometry. 25 (19), 2837-2843 (2011).</li><li>Wang, H., Liu, J., Cooks, R. 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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sceletium-a+review+update.">Sceletium-a review update.</a> Journal of Ethnopharmacology. 119 (3), 653-663 (2008).</li><li>Terburg, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+effects+of+Sceletium+tortuosum+(Zembrin),+a+dual+5-HT+reuptake+and+PDE4+inhibitor,+in+the+human+amygdala+and+its+connection+to+the+hypothalamus.">Acute effects of Sceletium tortuosum (Zembrin), a dual 5-HT reuptake and PDE4 inhibitor, in the human amygdala and its connection to the hypothalamus.</a> Neuropsychopharmacology. 38 (13), 2708-2716 (2013).</li><li>Coetzee, D. D., L&#243;pez, V., Smith, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-mesembrine+Sceletium+extract+(TrimesemineTM)+is+a+monoamine+releasing+agent,+rather+than+only+a+selective+serotonin+reuptake+inhibitor.">High-mesembrine Sceletium extract (TrimesemineTM) is a monoamine releasing agent, rather than only a selective serotonin reuptake inhibitor.</a> Journal of Ethnopharmacology. 177, 111-116 (2016).</li><li>Shikanga, E. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+permeation+of+mesembrine+alkaloids+from+Sceletium+tortuosum+across+porcine+buccal,+sublingual,+and+intestinal+mucosa.">In vitro permeation of mesembrine alkaloids from Sceletium tortuosum across porcine buccal, sublingual, and intestinal mucosa.</a> Planta Medica. 78 (3), 260-268 (2012).</li><li>Pulliam, C. J., Bain, R. M., Wiley, J. S., Ouyang, Z., Cooks, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass+spectrometry+in+the+home+and+garden.">Mass spectrometry in the home and garden.</a> Journal of The American Society for Mass Spectrometry. 26 (2), 224-230 (2015).</li><li>Lawton, Z. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analytical+validation+of+a+portable+mass+spectrometer+featuring+interchangeable,+ambient+ionization+sources.">Analytical validation of a portable mass spectrometer featuring interchangeable, ambient ionization sources.</a> Journal of the American Society for Mass Spectrometry. 28 (6), 1048-1059 (2017).</li><li>. GNPS Available from: <a target="_blank" href="https://gnps.ucsd.edu/">https://gnps.ucsd.edu/</a> (2018)</li><li>Chambers, M. 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The successful use of this protocol relies on the optimization of various steps for the plant species, tissue type, and target compound(s) of interest. The parameters described in the protocol provide a good starting point. The following experimental decisions need to be made and tested: whether or not to use (1) cut or uncut tissue and (2) solvent or no solvent, (3) what solvent to use and in what volume, (4) what the distance of tissue from the ion inlet should be, and (5) the voltage amplitude. The goal of optimizatio...

Plants contain a wide range of small molecules with diverse chemical properties. MS is a powerful technique for analyzing plant compounds because it can provide elemental compositions with a high sensitivity and specificity for the detection and identification of metabolites1. Most commonly, MS is performed on solvent-extracted samples, which are separated by chromatography prior to the MS analysis1. However, the use of liquid chromatography (LC) requires lengthy analysis times and is often associated with an extensive sample preparation1. In contrast, direct chemical analysis of intact tissues that circumvents chromatography is a very rapid technique, requiring minimal sample preparation2. Thus, in instances where chromatographic steps can be forgone, a direct chemical analysis can be highly advantageous.
Typical LC-MS for natural products and metabolomics research relies on lengthy bulk extractions of dried or frozen plant materials containing multiple tissues and cell types3. Alternatively, direct chemical analysis, such as the MS detection of metabolites from plant tissue, can isolate cell types and avoid preparation artifacts4. Leaf spray MS, also referred to as tissue-spray5,6, is a direct ambient ionization MS technique, which requires essentially no sample preparation5,7. Leaf spray MS is closely related to paper spray MS, an ambient ionization technique with characteristics of electrospray ionization that allows for the detection of analytes that are deposited onto paper7. In spite of the name, leaf spray MS is applicable to various types of plant tissues, not just leaves, and has been demonstrated on fruit, seeds, roots, floral tissues, and tubers, among others6,8,9,10,11,12. The technique facilitates the ionization of endogenous phytochemicals directly from the plant materials into the mass spectrometer for detection8. Leaf spray MS can also provide information about the spatial distribution of chemicals in different tissue types in plants13. When leaf spray MS is compared with solvent extraction and LC-MS, the results suggest leaf spray MS allows for the rapid detection of surface metabolites from unique cell types such as trichomes13. Figure 1 illustrates the leaf spray MS experimental set-up. Direct electrospray ionization occurs after only minor source modifications. A high voltage is applied to the plant tissue via a metal clamp, producing a spray of highly charged droplets forming a Taylor cone that carries the ions to the ion inlet of the MS. Electrospray ionization occurs from the natural liquid of the plant or from the solvent applied to the plant surface. A pointed tip on the tissue facilitates the electrospray and can be naturally occurring or created by cutting.
Leaf spray MS is a fast method for the qualitative and semi-quantitative analysis of intact plant tissues that have found utility for a wide variety of applications. For example, the technique has been used to detect endogenous compounds to distinguish related species, and even to evaluate changes in the same species grown under different conditions. Previous studies have shown this approach by measuring metabolites in beautyberry (Callicarpa L.)12 and American ginseng (Panax quinquefolium L.)6. In the latter example, ginsenosides, amino acids, and oligosaccharides could be detected after wetting raw ginseng tissue. Wild and cultivated American ginseng were differentiated from tuber slices6. Ginseng tuber integrity was preserved succeeding leaf spray MS, which allowed for a subsequent morphological and microscopic inspection6. Furthermore, exogenous compounds on plant samples can also be detected. A number of pesticides (acetamiprid, diphenylamine, imazalil, linuron, and thiabendazole) have been detected on peel or pulp of fruits and vegetables9. While these studies and many others have shown the utility of leaf spray MS for various specific purposes, a detailed protocol has not been previously reported.
Here, the protocol description will not focus on the optimization of the method for a specific tissue or compound. Rather, the detection of mesembrine alkaloids from Sceletium tortuosum (L.) N.E.Br. (Aizoaceae) is used as an example to discuss the necessary optimization measures that should be taken when setting up a leaf spray MS experiment for a species, tissue, or compound(s) for the first time. S. tortuosum is a succulent endemic to the semi-arid Karroo region of South Africa. A traditional medicine of the San and Khoi Khoi peoples, it was used for appetite and thirst suppression as well as for its psychotropic and analgesic effects14,15. Currently, standardized extracts are used for the treatment of neuropsychiatric and neuropsychological disorders16,17. The primary compounds of interest include the alkaloid mesembrine and its derivatives, many of which are also found in related Sceletium species15. Both wild and cultivated populations of S. tortuosum have variable concentrations of mesembrine alkaloids, thus presenting a quality control challenge18. A method for the rapid detection of mesembrine alkaloids, such as leaf spray MS, may be useful in monitoring Sceletium products. Because previously, there had been no detailed visual experimental protocol for the leaf spray MS technique, we will illustrate the method using the example of S. tortuosum, and the following is described: the modification of a nanospray source, the selection and preparation of the plant tissues, the acquisition of the data, the interpretation of the results, and the optimization of the MS parameters.

<table border="0" cellpadding="0" cellspacing="0" width="880">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Conn Pin</td>
			<td style="width:139px;">Digi-Key elctronics&#160;</td>
			<td style="width:119px;">WM2563CT-ND</td>
			<td style="width:385px;">pin will insert into Thermo Scientific&#160; source to provide voltage&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">small clamp</td>
			<td style="width:139px;">Digi-Key elctronics&#160;</td>
			<td>314-1018-ND</td>
			<td>CLIP MICRO ALLIGATOR COPPER 5A</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">large clamp</td>
			<td style="width:139px;">Digi-Key elctronics&#160;</td>
			<td>290-1951-ND</td>
			<td>ALLIGATOR CLIP NARROW NICKLE 5A</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Heat shrink</td>
			<td style="width:139px;">Digi-Key elctronics&#160;</td>
			<td style="width:119px;">Q2Z1-KIT-ND</td>
			<td>to cover soldering joints&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">NSI source Nanospray Ion Source</td>
			<td style="width:139px;">Thermo scientific</td>
			<td style="width:119px;">NA</td>
			<td>Another brand will work if you are not using a Thermo instrument</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:237px;">Q Exactive- hybrid quadrupole Orbitrap</td>
			<td style="width:139px;">Thermo scientific</td>
			<td style="width:119px;">NA</td>
			<td>Another brand will work if you are not using a Thermo instrument</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Tune Software</td>
			<td style="width:139px;">Thermo scientific</td>
			<td style="width:119px;"></td>
			<td>Another brand will work if you are not using a Thermo instrument</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Xcalibur Software</td>
			<td style="width:139px;">Thermo scientific</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Plant of interest - S. tortousum</td>
			<td style="width:139px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

leaf spray mass spectrometry: a rapid ambient ionization technique to directly assess metabolites from plant tissues

Plants produce thousands of small molecules that are diverse in their chemical properties. Mass spectrometry (MS) is a powerful technique for analyzing plant metabolites because it provides molecular weights with high sensitivity and specificity. Leaf spray MS is an ambient ionization technique where plant tissue is used for direct chemical analysis via electrospray, eliminating chromatography from the process. This approach to sampling metabolites allows for a wide range of chemical classes to be detected simultaneously from intact plant tissues, minimizing the amount of sample preparation needed. When used with a high-resolution, accurate mass MS, leaf spray MS facilitates the rapid detection of metabolites of interest. It is also possible to collect tandem mass fragmentation data with this technique to facilitate a compound identification. The combination of accurate mass measurements and fragmentation is beneficial in confirming compound identities. The leaf spray MS technique requires only minor modifications to a nanospray ionization source and is a useful tool to further expand the capabilities of a mass spectrometer. Here, fresh leaf tissue from Sceletium tortuosum (Aizoaceae), a traditional medicinal plant from South Africa, is analyzed; numerous mesembrine alkaloids are detected with leaf spray MS.

At 10 weeks post-germination, freshly collected greenhouse-grown S. tortuosum leaves were analyzed by leaf spray MS. The experimental workflow for detecting metabolites from S. tortuosum leaves using leaf spray MS is illustrated in Figure 2. A leaf was selected, cut into a thin strip with a tapered end to form a point, and clamped with the leaf spray MS wire clamp apparatus. The plant tissue was positioned ~30 mm from the ion inlet and in li...

Watch this Scientific Journal Video about Leaf Spray Mass Spectrometry: A Rapid Ambient Ionization Technique to Directly Assess Metabolites from Plant Tissues at JoVE.com

Leaf Spray Mass Spectrometry: A Rapid Ambient Ionization Technique to Directly Assess Metabolites from Plant Tissues

Leaf spray mass spectrometry is a direct chemical analysis technique that minimizes the sample preparation and eliminates chromatography, allowing for the rapid detection of small molecules from plant tissues.

Plants produce thousands of small molecules that are diverse in their chemical properties. Mass spectrometry (MS) is a powerful technique for analyzing ...

Leaf spray MS is a fast method for qualitative and semi-quantitative analysis of intact plant tissues that has a wide variety of applications. The main advantage of this technique is plant tissue can be analyzed directly with essentially no sample preparation. Although this method provides good insights into mesembrine alkaloids from Sceletium, it can also be used for analysis of various other plant species and exogenous chemicals such as pesticides.Individuals new to this method may find it challenging because it is necessary to optimize the protocol for each plant species, tissue type, and compound of interest. Use a modified nano spray source for leaf spray MS.As no fluidic components are necessary for leaf spray MS, modify the source by removing the liquid chromatography probe from the source. The leaf spray mass spectrometry wire that will apply voltage to the plant tissue has a pin that has been soldered to the wire on one end and a clamp that has been soldered on the other end.If the system has recently been in use, allow it to cool to the touch before removing any alternative source in the sweep cone. Then attach the nano spray leaf spray MS source. Finally, create a tune file and a method file as described in the text protocol.Always wear gloves and do not use plant tissue that has been handled with bare hands. Otherwise, contaminant ions such as polyethylene glycol will dominate the spectra. Bring the plant tissues for the analysis to the same room as the mass spectrometry system to allow for a rapid sampling.For plant tissue that does not have a naturally pointed tip, use a razor blade on a glass slide to cut a tapered point. Cut Sceletium tortuosum leaves at 10 weeks post germination into thin strips each with a tapered end to form a point. Use forceps to gently select the plant tissue at the end that will be clamped.Holding the tissue with forceps, carefully transfer it to the clamp. Adjust the flexible arm and the wire with the clamp to position the tissue in line with the MS inlet. Refer to the text for the optimal distance from the ion inlet for each specific mass spectrometer.If the first attempts produce a low signal intensity, the plant tissue can be moved closer to the ion inlet. Load the method file. Name the data file and set the file storage location.Then turn on the MS system by clicking play and click on start to begin acquiring data. Apply a solvent using a pipette with a gel loading tip to maximize the distance between the high voltage and the user's hands for protection. Acquire data as long as the signal persists or until the adequate spectra have been collected, typically 30 to 60 seconds.Stop the data acquisition and pause the MS system. Following acquisition, remove the tissue and wash the clamp with 100%methanol in a lint-free wipe. Open the data file and visually inspect the base peak mass chronogram.Check that the signal intensity is approximately 10 million to 500 million. Make a new method file with an inclusion list of M over Z out to four decimal places. Click on global lists and inclusion.Under properties of PRM, select the fragmentation energy and other tandem MS parameters. To obtain the tandem MS data for mesembrine alkaloids, fragment the ions at 276.1583 M over Z, 290.1742 M over Z, and 292.1897 M over Z at a normalized collision energy of 35. Next, load the tandem MS method file and a named data file.Turn on the MS system and start acquiring data adding a solvent if necessary. When adequate spectra have been collected, typically after 30 to 60 seconds, stop the acquisition. Perform data analysis as described in the text protocol.Representative results of metabolic profiling of Sceletium tortuosum by leaf spray MS and positive ionization are shown with the inset displaying 260 to 295 M over Z.In this study, a total of nine mesembrine alkaloids were detected by leaf spray MS and puditive identifications were made. The leaf spray MS total ion count mass chronogram represents a successful leaf spray MS experiment where a stable signal was maintained for the full duration of the acquisition. For each peak, the top number is the time in minutes and the bottom is the M over Z.Tandem mass spectra were collected in positive ionization made from a Sceletium tortuosum cut leaf with leaf spray MS.Puditive identifications of the alkaloids were made from accurate mass and mass fragmentation for mesembrine M and mesembrenone M isomers, mesembrine and mesembrine M dihydro.After watching this video, you should have a good understanding of how to use leaf spray MS to detect plant metabolites. Leaf spray MS has been used to detect endogenous compounds as well as exogenous compounds such as pesticides.

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Research

JoVE Journal

Biochemistry

Experimental Design for Laser Microdissection RNA-Seq: Lessons from an Analysis of Maize Leaf Development

Genes with important roles in development frequently have spatially and/or temporally restricted expression patterns. Often these gene transcripts are not detected or are not identified as differentially expressed (DE) in transcriptomic analyses of whole plant organs. Laser Microdissection RNA-Seq (LM RNA-Seq) is a powerful tool to identify genes that are DE in specific developmental domains. However, the choice of cellular domains to microdissect and compare, and the accuracy of the microdissections are crucial to the success of the experiments. Here, two examples illustrate design considerations for transcriptomics experiments; a LM RNA-seq analysis to identify genes that are DE along the maize leaf proximal-distal axis, and a second experiment to identify genes that are DE in liguleless1-R (lg1-R) mutants compared to wild-type. Key elements that contributed to the success of these experiments were detailed histological and in situ hybridization analyses of the region to be analyzed, selection of leaf primordia at equivalent developmental stages, the use of morphological landmarks to select regions for microdissection, and microdissection of precisely measured domains. This paper provides a detailed protocol for the analysis of developmental domains by LM RNA-Seq. The data presented here illustrate how the region selected for microdissection will affect the results obtained.

Many developmentally important genes have cell- or tissue-specific expression patterns. This paper describes LM RNA-seq experiments to identify genes that are differentially expressed at the maize leaf blade-sheath boundary and in lg1-R mutants compared to wild-type. The experimental considerations discussed here apply to transcriptomic analyses of other developmental phenomena.

Genes with important roles in development frequently have spatially and/or temporally restricted expression patterns. Often these gene transcripts are not ...

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. Hydrogen-Deuterium Exchange (HDX) measured at rapid time scales is uniquely suited to detect structures and hydrogen bonding networks that are briefly populated, allowing for the characterization of transient conformers in native ensembles. Coupling of HDX to mass spectrometry offers several key advantages, including high sensitivity, low sample consumption and no restriction on protein size. This technique has advanced greatly in the last several decades, including the ability to monitor HDX labeling times on the millisecond time scale. In addition, by incorporating the HDX workflow onto a microfluidic platform housing an acidic protease microreactor, we are able to localize dynamic properties at the peptide level. In this study, Time-Resolved ElectroSpray Ionization Mass Spectrometry (TRESI-MS) coupled to HDX was used to provide a detailed picture of residual structure in the tau protein, as well as the conformational shifts induced upon hyperphosphorylation.

Conformational flexibility plays a critical role in protein function. Herein, we describe the use of time-resolved electrospray ionization mass spectrometry coupled to hydrogen-deuterium exchange for probing the rapid structural changes that drive function in ordered and disordered proteins.

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological processes. Affinity purification coupled to mass spectrometry (AP-MS) has become the method of choice for identifying protein-protein interactions. However, conventional AP-MS studies provide information on protein interactions, but the organizational information is lost. To address this issue, we developed a strategy to unravel the distinct functional assemblies a protein might be involved in, by resolving affinity-purified protein complexes prior to their characterization by mass spectrometry. Protein complexes isolated through affinity purification of a bait protein using an epitope tag and competitive elution are separated through blue native electrophoresis. Comparison of protein migration profiles through correlation profiling using quantitative mass spectrometry allows assignment of interacting proteins to distinct molecular entities. This method is able to resolve protein complexes of close molecular weights that might not be resolved by traditional chromatographic techniques such as gel filtration. With little more work than conventional AP-geLC-MS/MS, we demonstrate this strategy may in many cases be adequate for obtaining protein complex topological information concomitantly to identifying protein interactions.

Here we present protocols for affinity purification of protein complexes and their separation by blue native PAGE, followed by protein correlation profiling using label free quantitative mass spectrometry. This method is useful to resolve interactomes into distinct protein complexes.

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological ...

A Simple Fractionated Extraction Method for the Comprehensive Analysis of Metabolites, Lipids, and Proteins from a Single Sample

Understanding of complex biological systems requires the measurement, analysis and integration of multiple compound classes of the living cell, usually determined by transcriptomic, proteomic, metabolomics and lipidomic measurements. In this protocol, we introduce a simple method for the reproducible extraction of metabolites, lipids and proteins from biological tissues using a single aliquot per sample. The extraction method is based on a methyl tert-butyl ether: methanol: water system for liquid: liquid partitioning of hydrophobic and polar metabolites into two immiscible phases along with the precipitation of proteins and other macromolecules as a solid pellet. This method, therefore, provides three different fractions of specific molecular composition, which are fully compatible with common high throughput 'omics' technologies such as liquid chromatography (LC) or gas chromatography (GC) coupled to mass spectrometers. Even though the method was initially developed for the analysis of different plant tissue samples, it has proved to be fully compatible for the extraction and analysis of biological samples from systems as diverse as algae, insects, and mammalian tissues and cell cultures.

A protocol for comprehensive extraction of lipids, metabolites and proteins from biological tissues using one sample is presented.

Understanding of complex biological systems requires the measurement, analysis and integration of multiple compound classes of the living cell, usually ...

Optimal Preparation of Formalin Fixed Samples for Peptide Based Matrix Assisted Laser Desorption/Ionization Mass Spectrometry Imaging Workflows

The use of matrix-assisted laser desorption/ionization, mass spectrometry imaging (MALDI MSI) has rapidly expanded, since this technique analyzes a host of biomolecules from drugs and lipids to N-glycans. Although various sample preparation techniques exist, detecting peptides from formaldehyde preserved tissues remains one of the most difficult challenges for this type of mass spectrometric analysis. For this reason, we have created and optimized a robust methodology that preserves the spatial information contained within the sample, while eliciting the greatest number of ionizable peptides. We have also aimed to achieve this in a cost effective and simple way, thereby eliminating potential bias or preparation error, which can occur when using automated instrumentation. The end result is a reproducible and inexpensive protocol.

This protocol describes a reproducible and reliable method for the sublimation-based preparation of formalin fixed tissue destined for imaging mass spectrometry.

The use of matrix-assisted laser desorption/ionization, mass spectrometry imaging (MALDI MSI) has rapidly expanded, since this technique analyzes a host ...

Detection of Protein Ubiquitination Sites by Peptide Enrichment and Mass Spectrometry

The posttranslational modification of proteins by the small protein ubiquitin is involved in many cellular events. After tryptic digestion of ubiquitinated proteins, peptides with a diglycine remnant conjugated to the epsilon amino group of lysine (&#39;K-&#949;-diglycine&#39; or simply &#39;diGly&#39;) can be used to track back the original modification site. Efficient immunopurification of diGly peptides combined with sensitive detection by mass spectrometry has resulted in a huge increase in the number of ubiquitination sites identified up to date. We have made several improvements to this workflow, including offline high pH reverse-phase fractionation of peptides prior to the enrichment procedure, and the inclusion of more advanced peptide fragmentation settings in the ion routing multipole. Also, more efficient cleanup of the sample using a filter-based plug in order to retain the antibody beads results in a greater specificity for diGly peptides. These improvements result in the routine detection of more than 23,000 diGly peptides from human cervical cancer cells (HeLa) cell lysates upon proteasome inhibition in the cell. We show the efficacy of this strategy for in-depth analysis of the ubiquitinome profiles of several different cell types and of in vivo samples, such as brain tissue. This study presents an original addition to the toolbox for protein ubiquitination analysis to uncover the deep cellular ubiquitinome.

We present a method for the purification, detection, and identification of diGly peptides that originate from ubiquitinated proteins from complex biological samples. The presented method is reproducible, robust, and outperforms published methods with respect to the level of depth of the ubiquitinome analysis.

The posttranslational modification of proteins by the small protein ubiquitin is involved in many cellular events. After tryptic digestion of ...

Sample Preparation for Probe Electrospray Ionization Mass Spectrometry

Mass spectrometry (MS) is a powerful tool in analytical chemistry because it provides very accurate information about molecules, such as mass-to-charge ratios (m/z), which are useful to deduce molecular weights and structures. While it is essentially a destructive analytical method, recent advancements in the ambient ionization technique have enabled us to acquire data while leaving tissue in a relatively intact state in terms of integrity. Probe electrospray ionization (PESI) is a so-called direct method because it does not require complex and time-consuming pretreatment of samples. A fine needle serves as a sample picker, as well as an ionization emitter. Based on the very sharp and fine property of the probe tip, destruction of the samples is minimal, allowing us to acquire the real-time molecular information from living things in situ. Herein, we introduce three applications of PESI-MS technique that will be useful for biomedical research and development. One involves the application to solid tissue, which is the basic application of this technique for the medical diagnosis. As this technique requires only 10 mg of the sample, it may be very useful in the routine clinical settings. The second application is for in vitro medical diagnostics where human blood serum is measured. The ability to measure fluid samples is also valuable in various biological experiments where a sufficient volume of sample for conventional analytical techniques cannot be provided. The third application leans toward the direct application of probe needles in living animals, where we can obtain real-time dynamics of metabolites or drugs in specific organs. In each application, we can infer the molecules that have been detected by MS or use artificial intelligence to obtain a medical diagnosis.

This article introduces sample preparation methods for a unique real-time analytical method based on the ambient mass spectrometry. This method lets us perform real-time analysis of the biological molecules in vivo without any special pretreatments.

Mass spectrometry (MS) is a powerful tool in analytical chemistry because it provides very accurate information about molecules, such as mass-to-charge ...

Isolation of Histone from Sorghum Leaf Tissue for Top Down Mass Spectrometry Profiling of Potential Epigenetic Markers

Histones belong to a family of highly conserved proteins in eukaryotes. They pack DNA into nucleosomes as functional units of chromatin. Post-translational modifications (PTMs) of histones, which are highly dynamic and can be added or removed by enzymes, play critical roles in regulating gene expression. In plants, epigenetic factors, including histone PTMs, are related to their adaptive responses to the environment. Understanding the molecular mechanisms of epigenetic control can bring unprecedented opportunities for innovative bioengineering solutions. Herein, we describe a protocol to isolate the nuclei and purify histones from sorghum leaf tissue. The extracted histones can be analyzed in their intact forms by top-down mass spectrometry (MS) coupled with online reversed-phase (RP) liquid chromatography (LC). Combinations and stoichiometry of multiple PTMs on the same histone proteoform can be readily identified. In addition, histone tail clipping can be detected using the top-down LC-MS workflow, thus, yielding the global PTM profile of core histones (H4, H2A, H2B, H3). We have applied this protocol previously to profile histone PTMs from sorghum leaf tissue collected from a large-scale field study, aimed at identifying epigenetic markers of drought resistance. The protocol could potentially be adapted and optimized for chromatin immunoprecipitation-sequencing (ChIP-seq), or for studying histone PTMs in similar plants.

The protocol has been developed to effectively extract intact histones from sorghum leaf materials for profiling of histone post-translational modifications that can serve as potential epigenetic markers to aid engineering drought resistant crops.

Histones belong to a family of highly conserved proteins in eukaryotes. They pack DNA into nucleosomes as functional units of chromatin. ...

Semi-Quantitative Analysis of Peptidoglycan by Liquid Chromatography Mass Spectrometry and Bioinformatics

Peptidoglycan is an important component of bacterial cell walls and a common cellular target for antimicrobials. Although aspects of peptidoglycan structure are fairly conserved across all bacteria, there is also considerable variation between Gram-positives/negatives and between species. In addition, there are numerous known variations, modifications, or adaptations to the peptidoglycan that can occur within a bacterial species in response to growth phase and/or environmental stimuli. These variations produce a highly dynamic structure that is known to participate in many cellular functions, including growth/division, antibiotic resistance, and host defense avoidance. To understand the variation within peptidoglycan, the overall structure must be broken down into its constitutive parts (known as muropeptides) and assessed for overall cellular composition. Peptidoglycomics uses advanced mass spectrometry combined with high-powered bioinformatic data analysis to examine peptidoglycan composition in fine detail. The following protocol describes the purification of peptidoglycan from bacterial cultures, the acquisition of muropeptide intensity data through a liquid chromatograph&#8212;mass spectrometer, and the differential analysis of peptidoglycan composition using bioinformatics.

This protocol covers a detailed analysis of peptidoglycan composition using liquid chromatography mass spectrometry coupled with advanced feature extraction and bioinformatic analysis software.

Peptidoglycan is an important component of bacterial cell walls and a common cellular target for antimicrobials. Although aspects of peptidoglycan ...

Quantitative Metabolomics of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

Metabolomics is a methodology used for the identification and quantification of many low-molecular-weight intermediates and products of metabolism within a cell, tissue, organ, biological fluid, or organism. Metabolomics traditionally focuses on water-soluble metabolites. The water-soluble metabolome is the final product of a complex cellular network that integrates various genomic, epigenomic, transcriptomic, proteomic, and environmental factors. Hence, the metabolomic analysis directly assesses the outcome of the action for all these factors in a plethora of biological processes within various organisms. One of these organisms is the budding yeast Saccharomyces cerevisiae, a unicellular eukaryote with the fully sequenced genome. Because S. cerevisiae is amenable to comprehensive molecular analyses, it is used as a model for dissecting mechanisms underlying many biological processes within the eukaryotic cell. A versatile analytical method for the robust, sensitive, and accurate quantitative assessment of the water-soluble metabolome would provide the essential methodology for dissecting these mechanisms. Here we present a protocol for the optimized conditions of metabolic activity quenching in and water-soluble metabolite extraction from S. cerevisiae cells. The protocol also describes the use of liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for the quantitative analysis of the extracted water-soluble metabolites. The LC-MS/MS method of non-targeted metabolomics described here is versatile and robust. It enables the identification and quantification of more than 370 water-soluble metabolites with diverse structural, physical, and chemical properties, including different structural isomers and stereoisomeric forms of these metabolites. These metabolites include various energy carrier molecules, nucleotides, amino acids, monosaccharides, intermediates of glycolysis, and tricarboxylic cycle intermediates. The LC-MS/MS method of non-targeted metabolomics is sensitive and allows the identification and quantitation of some water-soluble metabolites at concentrations as low as 0.05 pmol/&#181;L. The method has been successfully used for assessing water-soluble metabolomes of wild-type and mutant yeast cells cultured under different conditions.

Quantitative Metabolomics of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

We present a protocol for the identification and quantitation of major classes of water-soluble metabolites in the yeast Saccharomyces cerevisiae. The described method is versatile, robust, and sensitive. It allows the separation of structural isomers and stereoisomeric forms of water-soluble metabolites from each other.

Metabolomics is a methodology used for the identification and quantification of many low-molecular-weight intermediates and products of metabolism within ...