Name: isolation of histone from sorghum leaf tissue for top down mass spectrometry profiling of potential epigenetic markers
Uploaded: 2021-03-04T13:00:00
Description: Watch this Scientific Journal Video about Isolation of Histone from Sorghum Leaf Tissue for Top Down Mass Spectrometry Profiling of Potential Epigenetic Markers at JoVE.com

We thank Ronald Moore and Thomas Fillmore for helping with mass spectrometry experiments, and Matthew Monroe for data deposition. This research was funded by grants from US Department of Energy (DOE) Biological and Environmental Research through the Epigenetic Control of Drought Response in Sorghum (EPICON) project under award number DE-SC0014081, from the US Department of Agriculture (USDA; CRIS 2030-21430-008-00D), and through the Joint BioEnergy Institute (JBEI), a facility sponsored by DOE (Contract DE-AC02-05CH11231) between Lawrence Berkeley National Laboratory and DOE. The research was performed using Environmental Molecular Sciences Laboratory (EMSL) (grid.436923.9), a DOE Office of Science User Facility sponsored by the Office of Biological and Environmental Research.

1. Preparing sorghum leaf material
NOTE: The sorghum plants were grown in soil in the field in Parlier, CA.
<ol>
	<li>Collect sorghum leaves from plants into 50 mL centrifuge tubes and immediately freeze the tube in liquid nitrogen. Collect leaf tissue by tearing off the third and fourth fully emerged leaf from the primary tiller. 
	NOTE: More details of field condition, sample growth, and collection can be found in the published report18.</li>
	<li>Grind leaves ...

<ol>
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The presented protocol describes how to extract histones from sorghum leaf (or more generally plant leaf) samples. The average histone yield is expected to be 2&#8211;20 &#181;g per 4&#8211;5 g sorghum leaf material. The materials are sufficiently pure for the downstream histone analysis by LC-MS (mostly histones with ~20% ribosomal protein contamination). Lower yield may be obtained due to sample variations, or potential mishandling/failures throughout the protocol. Maintaining the integrity of the nuclei before the nuc...

The increasing severity and frequency of drought is expected to affect productivity of cereal crops1,2. Sorghum is a cereal food and energy crop known for its exceptional ability to withstand water-limiting conditions3,4. We are pursuing mechanistic understanding of the interplay between drought stress, plant development, and epigenetics of sorghum [Sorghum bicolor (L.) Moench] plants. Our previous work has demonstrated strong connections between plant and rhizosphere microbiome in drought acclimation and responses at the molecular level5,6,7. This research will pave the way for utilizing epigenetic engineering in adapting crops to future climate scenarios. As a part of the efforts in understanding epigenetics, we aim to study protein markers that impact gene expression within the plant organism.
Histones belong to a highly conserved family of proteins in eukaryotes that pack DNA into nucleosomes as fundamental units of chromatin. Post-translational modifications (PTMs) of histones are dynamically regulated to control chromatin structure and influence gene expression. Like other epigenetic factors, including DNA methylation, histone PTMs play important roles in many biological processes8,9. Antibody-based assays such as western blots have widely been used to identify and quantify histone PTMs. In addition, the interaction of histone PTMs and DNA can be effectively probed by Chromatin immunoprecipitation &#8211; sequencing (ChIP-seq)10. In ChIP-seq, chromatin with specific targeted histone PTM is enriched by antibodies against that specific PTM. Then, the DNA fragments can be released from the enriched chromatin and sequenced. Regions of genes that interact with the targeted histone PTM are revealed. However, all these experiments heavily rely on high quality antibodies. For some histone variants/homologs or combinations of PTMs, development of robust antibodies can be extremely challenging (especially for multiple PTMs). In addition, antibodies can only be developed if the targeted histone PTM is known.11 Therefore, alternative methods for untargeted, global profiling of histone PTMs are necessary.
Mass spectrometry (MS) is a complementary method to characterize histone PTMs, including unknown PTMs for which antibodies are not available11,12. The well-established &#8220;bottom-up&#8221; MS workflow uses proteases to digest proteins into small peptides prior to liquid chromatography (LC) separation and MS detection. Because histones have large numbers of basic residues (lysine and arginine), the trypsin digestion (protease specific to lysine and arginine) in the standard bottom-up workflow cuts the proteins into very short peptides. The short peptides are technically difficult to analyze by standard LC-MS, and do not preserve the information about the connectivity and stoichiometry of multiple PTMs. The use of other enzymes or chemical labeling to block lysines generates longer peptides that are more suitable for characterization of histone PTMs13,14.
Alternatively, the digestion step can be completely omitted. In this &#8220;top-down&#34; approach, intact protein ions are introduced into the MS by electrospray ionization (ESI) after online LC separation, yielding ions of the intact histone proteoforms. In addition, ions (i.e., proteoforms) of interest can be isolated and fragmented in the mass spectrometer to yield the sequence ions for identification and PTM localization. Hence, top-down MS has the advantage to preserve the proteoform-level information and capture the connectivity of multiple PTMs and terminal truncations on the same proteoform15,16. Top-down experiments can also provide quantitative information and offer insights of biomarkers at the intact protein level17. Herein, we describe a protocol to extract histone from sorghum leaf and analyze the intact histones by top-down LC-MS.
The example data shown in Figure 1 and Figure 2 are from sorghum leaf collected at week 2 after planting. Although variation of yield is expected, this protocol is generally agnostic to specific sample conditions. The same protocol has been successfully used for sorghum plant leaf tissue collected from 2, 3, 5, 8, 9, and 10 weeks after planting.

<table>
	<tbody>
		<tr>
			<td>Acetonitrile</td>
			<td>Fisher Chemical</td>
			<td>A955-4L</td>
			<td></td>
		</tr>
		<tr>
			<td>Dithiothreitol (DTT)</td>
			<td>Sigma</td>
			<td>43815-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA, 500mM Solution, pH 8.0</td>
			<td>EMD Millipore Corp</td>
			<td>324504-500mL</td>
			<td></td>
		</tr>
		<tr>
			<td>Formic Acid</td>
			<td>Thermo Scientific</td>
			<td>28905</td>
			<td></td>
		</tr>
		<tr>
			<td>Guanidine Hydrochloride</td>
			<td>Sigma</td>
			<td>G3272-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma</td>
			<td>M8266-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium phosphate, dibasic</td>
			<td>Sigma</td>
			<td>P3786-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease Inhibitor Cocktail, cOmplete tablets</td>
			<td>Roche</td>
			<td>5892791001</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium butyrate</td>
			<td>Sigma</td>
			<td>303410-5G</td>
			<td>Used for histone deacetylase inhibitor</td>
		</tr>
		<tr>
			<td>Sodium Chloride (NaCl)</td>
			<td>Sigma</td>
			<td>S1888</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Fluoride</td>
			<td>Sigma</td>
			<td>S7020-100G</td>
			<td>Used for phosphatase inhibitor</td>
		</tr>
		<tr>
			<td>Sodium Orthovanadate</td>
			<td>Sigma</td>
			<td>450243-10G</td>
			<td>Used for phosphatase inhibitor</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma</td>
			<td>S7903-5KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-HCl</td>
			<td>Fisher Scientific</td>
			<td>BP153-500 g</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>T9284-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Weak cation exchange resin, mesh 100-200 analytical (BioRex70)</td>
			<td>Bio-Rad</td>
			<td>142-5842</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposables</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Chromatography column (Bio-Spin)</td>
			<td>BIO-RAD</td>
			<td>732-6008</td>
			<td></td>
		</tr>
		<tr>
			<td>Mesh 100 filter cloth</td>
			<td>Millipore Sigma</td>
			<td>NY1H09000</td>
			<td>This is part of the Sigma kit (catalog # CELLYTPN1) for plant nuclei extraction. Similar filters with the same mesh size can be used.</td>
		</tr>
		<tr>
			<td>Micropipette tips (P20, P200, P1000)</td>
			<td>Sigma</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tube, 50mL/15mL, Centrifuge, Conical</td>
			<td>Genesee Scientific</td>
			<td>28-103</td>
			<td></td>
		</tr>
		<tr>
			<td>Tube, Microcentrifuge, 1.5/2 mL</td>
			<td>Sigma</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Analytical Balance</td>
			<td>Fisher Scientific</td>
			<td>01-912-401</td>
			<td></td>
		</tr>
		<tr>
			<td>Beakers (50mL &#8211; 2L)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge with cooling</td>
			<td>Fisher Scientific</td>
			<td>13-690-006</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipettes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Swinging-bucket centrifuge with cooling</td>
			<td>Fisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex</td>
			<td>Fisher Scientific</td>
			<td>50-728-002</td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath Sonicator</td>
			<td>Fisher Scientific</td>
			<td>15-336-120</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Following the protocol, the histones can be extracted and identified using the LC-MS analysis. The raw data and processed results are available at MassIVE (https://massive.ucsd.edu/) via accession: MSV000085770. Based on the TopPIC results from the representative sample (available also from MassIVE), we identified 303 histone proteoforms (106 H2A, 72 H2B, 103 H3, and 22 H4 proteoforms). Co-purified ribosomal proteoforms have also been detected, typically eluting early in the LC. They usually consist of ~20% of the identi...

Watch this Scientific Journal Video about Isolation of Histone from Sorghum Leaf Tissue for Top Down Mass Spectrometry Profiling of Potential Epigenetic Markers at JoVE.com

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

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. ...

Our protocol uncovers epigenetic markers for sorghum plant development and drought resistance. Such molecular understanding will help develop better solutions to adapt crops for extreme climates in the future. This protocol generates high purity histones from sorghum leaf tissue suitable for un-targeted profiling of post-translational modifications by mass spectrometry.Demonstrating the procedure will be Shadan Abdali and Tanya Winkler, post-bachelor research associates from EMSL. To begin, grind a few sorghum leaves with liquid nitrogen and store them in a 50 milliliter centrifuge tube at minus 80 degrees Celsius. Use approximately four grams of the leaf powder for histone analysis of each sample.Prepare extraction buffers 1, 2A, and 2B as described in the text manuscript and add a protease inhibitor tablet to extraction buffer one to make a final concentration of 0.2X. Then add 20 milliliters of the prepared extraction buffer one to the ground leaf powder and gently mix or vortex for 10 minutes. Using mesh 100, rinse the filtered material twice with two milliliters of extraction buffer one each time.Next, centrifuge the filtrate at 3, 000 times G for 10 minutes at four degrees Celsius in a swinging bucket rotor to pellet the cell debris and large subcellular organelles. Simultaneously, prepare extraction buffer 2A by adding protease inhibitor to a final concentration of 0.4X. Discard the supernatant without disturbing the pellet.Then resuspend the pellet in five milliliters of the prepared extraction buffer 2A. Incubate it on ice for 10 minutes with gentle mixing. Centrifuge the solution at 2, 100 times G for 15 minutes at four degrees Celsius in a swinging bucket rotor to pellet the debris and nuclei.Decant the supernatant carefully without disturbing the pellet. Prepare extraction buffer 2B by adding the protease inhibitors to a final concentration of 1X. Add five milliliters of this prepared buffer to the pellet obtained after the centrifugation.Centrifuge at 2, 100 times G for 15 minutes at four degrees Celsius in the swinging bucket rotor to pellet debris and nuclei. Simultaneously, prepare nuclei lysis buffer by adding a protease inhibitor tablet. Carefully decant the supernatant without disturbing the pellet and resuspend the pellet in 250 microliters of the lysis buffer.Vortex the solution for 15 seconds at maximum speed to homogenize it and resuspend the material. Then sonicate it for five minutes at four degrees Celsius and store it at minus 80 degrees Celsius. Thaw the frozen nuclei sample and add 750 microliters of 5%guanidine buffer.Then sonicate it for 15 minutes at four degrees Celsius. Transfer the sample to a two milliliter tube and spin it at 10, 000 times G for 10 minutes at four degrees Celsius. Simultaneously, prepare an ion exchange chromatography column by rinsing it with two milliliters of acetonitrile and four milliliters of water to minimize contamination on the surface.Load approximately 200 to 300 microliters of weak cation exchange resin onto the chromatography column and let the resin settle. Wash the resin four times with guanidine buffer and place the tube and column on ice. Put the column on a two milliliter collection tube and load the supernatant from the prepared sample slowly onto the resin bed without disrupting the resin.As the solution is flowing through, load the eluent back to the top of the column six to eight times to allow maximum binding to the resin. Then load two milliliters of 5%guanidine buffer to wash the non-histone proteins off the column. Elute the histone proteins with one milliliter of 5%guanidine buffer and collect the eluent.Clean a three kilodalton cutoff spin filter with 500 microliters of wash solvent, then desalt the remaining eluent using the pre-cleaned spin filter. Major histone proteins were observed in specific regions of the LC-MS feature map indicating the success of the experiment. This representative map shows intact full-length histones in dashed boxes.The LC-MS feature map of H2B and 16 kilodalton H2A proteoforms shows that both have multiple homologs with similar sequences as noted by the unique product session numbers. Another group of H2A histones without the extended tails can be seen. The N-terminal acetylation, additional lysine acetylations, and methionine oxidations in H4 histone proteins can be observed by examining the mass differences in the LC-MS feature map shown here.There were two protein sequences identified for H3 histone proteins, H3.3 and H3.2. Electron transfer dissociation fragmentation was used to generate the fragmentation spectrum of the identified H3.2 proteoform. The precursor ions and the previous and next Ms1 spectra are shown here with their matched isotope peaks highlighted in purple.Post-translational modifications can be localized using the sequence coverage map. By comparing the relative abundance of the proteoforms, changes of truncated histone proteoforms specific to sample conditions were discovered. C-terminal truncation of H4 was observed only in weeks three and nine for some of the samples.For H3.2, N-terminal truncated proteoforms were generally more abundant in week 10. In contrast, C-terminal truncated H3.2 were seen in earlier time points. The H4 C-terminal truncated proteoforms were significantly more abundant in BTx642 than in RTx430.When attempting this protocol, pay close attention to the colors of the supernatant and the pellet. The color change help identify potential problems in the grinding or lysis of the chloroplast. We hope that this robust protocol for histone isolation from sorghum leaves will enable epigenetic research for other similar plants.

isolation-histone-from-sorghum-leaf-tissue-for-top-down-mass

Research

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Biochemistry

Sheathless Capillary Electrophoresis&#8211;Mass Spectrometry for Metabolic Profiling of Biological Samples

In metabolomics, a wide range of analytical techniques is used for the global profiling of (endogenous) metabolites in complex samples. In this paper, a protocol is presented for the analysis of anionic and cationic metabolites in biological samples by capillary electrophoresis&#8211;mass spectrometry (CE-MS). CE is well-suited for the analysis of highly polar and charged metabolites as compounds are separated on the basis of their charge-to-size ratio. A recently developed sheathless interfacing design, i.e., a porous tip interface, is used for coupling CE to electrospray ionization (ESI) MS. This interfacing approach allows the effective use of the intrinsically low-flow property of CE in combination with MS, resulting in nanomolar detection limits for a broad range of polar metabolite classes. The protocol presented here is based on employing a bare fused-silica capillary with a porous tip emitter at low-pH separation conditions for the analysis of a broad array of metabolite classes in biological samples. It is demonstrated that the same sheathless CE-MS method can be used for the profiling of cationic metabolites, including amino acids, nucleosides and small peptides, or anionic metabolites, including sugar phosphates, nucleotides and organic acids, by only switching the MS detection and separation voltage polarity. Highly information-rich metabolic profiles in various biological samples, such as urine, cerebrospinal fluid and extracts of the glioblastoma cell line, can be obtained by this protocol in less than 1 hr of CE-MS analysis.

A protocol for metabolic profiling of biological samples by capillary electrophoresis&#8211;mass spectrometry using a sheathless porous tip interface design is presented.

In metabolomics, a wide range of analytical techniques is used for the global profiling of (endogenous) metabolites in complex samples. In this paper, a ...

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 ...

Lipid Droplet Isolation for Quantitative Mass Spectrometry Analysis

Lipid droplets are vital to the replication of a variety of different pathogens, most prominently the Hepatitis C Virus (HCV), as the putative site of virion morphogenesis. Quantitative lipid droplet proteome analysis can be used to identify proteins that localize to or are displaced from lipid droplets under conditions such as virus infections. Here, we describe a protocol that has been successfully used to characterize the changes in the lipid droplet proteome following infection with HCV. We use Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) and thus label the complete proteome of one population of cells with &#34;heavy&#34; amino acids to quantitate the proteins by mass spectrometry. For lipid droplet isolation, the two cell populations (i.e.&#160;HCV-infected/&#34;light&#34; amino acids and uninfected control/&#34;heavy&#34; amino acids) are mixed 1:1 and lysed mechanically in hypotonic buffer. After removing the nuclei and cell debris by low speed centrifugation, lipid droplet-associated proteins are enriched by two subsequent ultracentrifugation steps followed by three washing steps in isotonic buffer. The purity of the lipid droplet fractions is analyzed by western blotting with antibodies recognizing different subcellular compartments. Lipid droplet-associated proteins are then separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by Coomassie staining. After tryptic digest, the peptides are quantified by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). Using this method, we identified proteins recruited to lipid droplets upon HCV infection that might represent pro- or antiviral host factors. Our method can be applied to a variety of different cells and culture conditions, such as infection with pathogens, environmental stress, or drug treatment.

Lipid droplets are important organelles for the replication of several pathogens, including the Hepatitis C Virus (HCV). We describe a method to isolate lipid droplets for quantitative mass spectrometry of associated proteins; it can be used under a variety of conditions, such as virus infection, environmental stress, or drug treatment.

Lipid droplets are vital to the replication of a variety of different pathogens, most prominently the Hepatitis C Virus (HCV), as the putative site of ...

Sample Preparation for Mass Spectrometry-based Identification of RNA-binding Regions

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most cases, the action of noncoding RNAs is mediated by proteins whose functions are in turn regulated by these interactions with noncoding RNAs. Consistent with this, a growing number of proteins involved in nuclear functions have been reported to bind RNA and in a few cases the RNA-binding regions of these proteins have been mapped, often through laborious, candidate-based methods.
Here, we report a detailed protocol to perform a high-throughput, proteome-wide unbiased identification of RNA-binding proteins and their RNA-binding regions. The methodology relies on the incorporation of a photoreactive uridine analog in the cellular RNA, followed by UV-mediated protein-RNA crosslinking, and mass spectrometry analyses to reveal RNA-crosslinked peptides within the proteome. Although we describe the procedure for mouse embryonic stem cells, the protocol should be easily adapted to a variety of cultured cells.

We describe a protocol to identify RNA-binding proteins and map their RNA-binding regions in live cells using UV-mediated photocrosslinking and mass spectrometry.

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most ...

Deep Proteome Profiling by Isobaric Labeling, Extensive Liquid Chromatography, Mass Spectrometry, and Software-assisted Quantification

Many exceptional advances have been made in mass spectrometry (MS)-based proteomics, with particular technical progress in liquid chromatography (LC) coupled to tandem mass spectrometry (LC-MS/MS) and isobaric labeling multiplexing capacity. Here, we introduce a deep-proteomics profiling protocol that combines 10-plex tandem mass tag (TMT) labeling with an extensive LC/LC-MS/MS platform, and post-MS computational interference correction to accurately quantitate whole proteomes. This protocol includes the following main steps: protein extraction and digestion, TMT labeling, 2-dimensional (2D) LC, high-resolution mass spectrometry, and computational data processing. Quality control steps are included for troubleshooting and evaluating experimental variation. More than 10,000 proteins in mammalian samples can be confidently quantitated with this protocol. This protocol can also be applied to the quantitation of post translational modifications with minor changes. This multiplexed, robust method provides a powerful tool for proteomic analysis in a variety of complex samples, including cell culture, animal tissues, and human clinical specimens.

We present a protocol to accurately quantitate proteins with isobaric labelling, extensive fractionation, bioinformatics tools, and quality control steps in combination with liquid chromatography interfaced to a high-resolution mass spectrometer.

Many exceptional advances have been made in mass spectrometry (MS)-based proteomics, with particular technical progress in liquid chromatography (LC) ...

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 ...

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.

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 ...

Nitropeptide Profiling and Identification Illustrated by Angiotensin II

Protein nitration is one of the most important post-translational modifications (PTM) on tyrosine residues and it can be induced by chemical actions of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in eukaryotic cells. Precise identification of nitration sites on proteins is crucial for understanding the physiological and pathological processes related to protein nitration, such as inflammation, aging, and cancer. Since the nitrated proteins are of low abundance in cells even under induced conditions, no universal and efficient methods have been developed for the profiling and identification of protein nitration sites. Here we describe a protocol for nitropeptide enrichment by using a chemical reduction reaction and biotin labeling, followed by high resolution mass spectrometry. In our method, nitropeptide derivatives can be identified with high accuracy. Our method exhibits two advantages compared to the previously reported methods. First, dimethyl labeling is used to block the primary amine on nitropeptides, which can be used to generate quantitative results. Second, a disulfide bond containing NHS-biotin reagent is used for the enrichment, which can be further reduced and alkylated to enhance the detection signal on a mass spectrometer. This protocol has been successfully applied to the model peptide Angiotensin II in the current paper.

Proteomic profiling of tyrosine-nitrated proteins has been a challenging technique due to the low abundance of the 3-nitrotyrosine modification. Here we describe a novel approach for nitropeptide enrichment and profiling by using Angiotensin II as the model. This method can be extended for other in vitro or in vivo systems.

Protein nitration is one of the most important post-translational modifications (PTM) on tyrosine residues and it can be induced by chemical actions 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 ...