R.B. was supported by the Searle Scholars Program, the W.W. Smith Foundation (C1404), and the March of Dimes Foundation (1-FY-15- 344). B.A.G acknowledges support from NIH grants R01GM110174 and NIH R01AI118891, as well as DOD grant BC123187P1. R.W.-T. was supported by NIH training grant T32GM008216.

1. Culture and Expansion of mESCs
NOTE: Mouse embryonic stem cells are easy to culture and can be quickly expanded to the large numbers required by biochemical experiments thanks to their fast cycling time. Healthy mESCs double every 12 h.
<ol>
	<li>Expand mESCs to the desired number on gelatinized plates in mESC medium (see below) in a tissue culture incubator kept at 37 &#176;C, 5% CO2, and &#62;95% humidity. 
	NOTE: Enough plates should be prepared for 3 - 5 biological replicates for the +4SU sample and the -4SU control. One 10-cm plate (i.e. 5 - 10 million cells) is more than sufficient for each biological replicate.</li>
	<li>The medium for mESCs is composed of Dulbecco&#39;s modified Eagle&#39;s medium (DMEM) supplemented with 15% fetal bovine serum (FBS), 2 mM L-glutamine, 0.5x penicillin/streptomycin, 1x non-essential amino acids, 100 &#181;M beta-mercaptoethanol, 1,000 U/mL leukemia inhibitory factor (LIF), 3 &#181;M CHIR99021, 1 &#181;M PD0325901.</li>
	<li>Before passaging mESCs, prepare the desired number of 10 cm plates.
	<ol>
		<li>Add 4 mL of 0.1% gelatin in water making sure that the entire surface of the plate is covered; incubate for 10 min at room temperature (RT); remove gelatin solution.</li>
	</ol>
	</li>
	<li>Passage cells when they reach ~10 million cells per plate, typically ~48 h following inoculation. 
	NOTE: As mESCs grow in dense colonies, plates should never be allowed to reach 100% confluency as observed for cell lines that grow in monolayers (e.g. HEK293 or 3T3 cells). An mESC plate should be passaged when ~50% confluent and definitely before the medium turns yellow.
	<ol>
		<li>To detach cells, wash plates with dense mESC cultures once in 1x phosphate buffered saline (PBS) supplemented with 2 mM EDTA, then add 0.05% trypsin dissolved in DMEM and return plate to incubator for 5 min (37 &#176;C, 5% CO2, and &#62;95% humidity).</li>
		<li>Remove plate from incubator and resuspend cells with equal volume of DMEM by pipetting up and down vigorously several times.</li>
		<li>Count cells using hemocytometer and ensure that cells form a single-cell suspension (i.e. no clusters), then centrifuge cell mixture 500 x g for 5 min at room temperature. Remove supernatant and resuspend cell pellet in 1 - 2 mL DMEM.</li>
		<li>Inoculate cells at a density of 10,000 cells/cm2 in mESC medium (i.e. ~800,000 cells per 10-cm plate) using the gelatin-coated plates prepared in step 1.3.1.</li>
	</ol>
	</li>
</ol>
2. Crosslink of Protein&#8211;RNA Interactions in Live Cells
NOTE: RNA-protein crosslinking is mediated by the photo-activatable ribonucleoside analog 4-thiouridine (4SU). 4SU has a longer absorbance maximum than endogenous nucleotides and can only be incorporated into RNA; therefore intermediate-wavelength UVB can be used to selectively crosslink RNA to proteins25,26. UVB treatment of 4SU-treated cells leads to covalent crosslinks between 4SU-containing RNA and amino acids, with a reported preference for Tyr, Trp, Met, Lys, and Cys27.
<ol>
	<li>Expand mESCs to the required number of 10-cm plates as described in step 1. 
	NOTE: Enough plates should be prepared for 3 - 5 biological replicates for the +4SU sample and the -4SU control. One 10-cm plate (i.e. 5 - 10 million cells) is sufficient for each biological replicate.</li>
	<li>Before the plates reach confluency, add 4SU directly into medium to a final concentration of 500 &#181;M. Leave -4SU control dishes untreated.</li>
	<li>Shake plates gently to distribute the 4SU homogeneously throughout the medium. Return them to the same tissue culture incubator for 2 h (37 &#176;C, 5% CO2, and &#62;95% humidity).</li>
	<li>Transfer plates to an ice bucket and discard medium.</li>
	<li>Rinse the plate gently with 5 mL of ice-cold PBS. 
	NOTE: Rinse gently or cells might detach.</li>
	<li>Add 2 mL of ice-cold PBS and place plates without lids in a crosslinker equipped with UVB-emitting bulbs (312 nm). Irradiate plates at an energy setting of 1 J/cm2. 
	NOTE: It is critical that plastic lids are removed or they might shield the cells from UV light and prevent crosslinking.</li>
	<li>Collect cells using cell lifters and transfer to ice-cold PBS in conical tubes. Centrifuge at 2,000 x g for 5 min, 4 &#176;C.</li>
	<li>Remove the supernatant and resuspend cell pellet in 5 mL ice-cold PBS with 2 mM EDTA and 0.2 mM phenylmethylsulfonyl fluoride (PMSF).</li>
	<li>Count cells using a hemocytometer. Expect 5 - 10 and 10 - 20 million cells from 10-cm and 15-cm plates respectively.</li>
	<li>Centrifuge at 2,000 x g for 5 min, 4 &#176;C. 
	NOTE: Pelleted cells can be immediately used for step 3 or they can be flash-frozen in liquid nitrogen and stored at -80 &#176;C indefinitely. This is a potential stopping point.</li>
</ol>
3. Isolation of Nuclei
NOTE: Nuclei are isolated to remove cytoplasmic proteins and increase coverage of nuclear proteins. This step can be replaced with other forms of cellular fractionation to study RBPs in different cellular compartments.
<ol>
	<li>Prepare ice cold Buffer A (10 mM Tris-HCl pH 7.9, 4 &#176;C, 1.5 mM MgCl2, 10 mM KCl). Use at least 2.5 mL per 10 million cells (as counted in step 2.9) for extraction. 
	NOTE: Stocks of Buffer A (e.g. a 500 mL bottle) can be prepared in advance and stored at 4 &#176;C for 6 months.
	<ol>
		<li>Prior to use, add 0.5 mM dithiothreitol (DTT) and 0.2 mM PMSF to the desired amount (2.5 mL per 10 million cells) of ice-cold buffer A.</li>
	</ol>
	</li>
	<li>Wash cells using 2 mL Buffer A per 10 million cells. Spin at 2,500 x g for 5 min at 4 &#176;C.</li>
	<li>Discard supernatant, add Buffer A supplemented with 0.2% of a nonionic, non-denaturing detergent, such as octyl-phenoxy-polyethoxy-ethanol (see the Table of Materials), 500 &#181;L per 10 million cells. Rotate for 5 min at 4 &#176;C.</li>
	<li>Centrifuge at 2,500 x g for 5 min.</li>
	<li>Remove the supernatant; the pellet comprises intact nuclei devoid of cytoplasm. 
	NOTE: Nuclei can be used immediately for step 4 or flash-frozen in liquid nitrogen and stored at -80 &#176;C indefinitely. This is a potential stopping point.</li>
</ol>
4. Lysis of Nuclei
NOTE: Crosslinked nuclei are lysed in a mass spectrometry-compatible buffer to release proteins and protein-RNA complexes.
<ol>
	<li>Add lysis buffer (9 M urea, 100 mM Tris-HCl pH 8.0, 24 &#176;C), 200 &#181;L per 10 million cells. Sonicate to shear chromatin using a 1/8&#34; probe at 50% amplitude setting for 5 - 10 s.</li>
	<li>Spin at 12,000 x g for 5 min and collect 175 &#181;L of supernatant to avoid the pellet. 
	NOTE: 100 &#181;L will be used for the next RBR-ID steps and the remaining 75 &#181;L of lysate can be stored at -80 &#176;C for later use and/or western blot analysis.</li>
	<li>Measure protein concentration via Bradford assay or similar technique28. Dilute protein concentration in the different samples to 1 mg/mL or the lowest sample concentration using appropriate amounts of lysis buffer. 
	NOTE: From the nuclei of 10 million mESCs expect 100 -&#160;200 &#181;g of protein.</li>
</ol>
5. Trypsin Digestion
NOTE: Proteins are digested to generate peptides suitable for bottom-up mass spectrometry (MS) analysis.
<ol>
	<li>Add DTT solution to nuclear lysate (5 mM final); incubate for 1 h at RT.</li>
	<li>Add iodoacetamide from fresh stock (14 mM final); incubate for 30 min at RT in the dark.</li>
	<li>Dilute lysate with 5 times volume of 50 mM NH4HCO3</li>
	<li>Add trypsin (&#181;g protein : &#181;g trypsin = 200 : 1); incubate at 37 &#176;C overnight.</li>
</ol>
6. Desalting of peptides
<ol>
	<li>Prepare one custom-made stage tip per sample.
	<ol>
		<li>6.1.1. Place a disk of solid phase extraction C18 material into the bottom of a 200 &#181;L pipette tip.</li>
		<li>Add 50 &#181;L of oligo R3 reversed phase resin on top of the C18 disk. Place stage tip inside centrifugation adaptor and the place tip with adaptor inside a 1.5 mL microfuge tube.</li>
		<li>Add 100 &#181;L 100% acetonitrile to tips to wash. Centrifuge at 1000 x g for 1 min to remove the solvent.</li>
		<li>Equilibrate with 50 &#181;L 0.1% trifluoroacetic acid (TFA). Centrifuge at 1000 x g for 1 min.</li>
	</ol>
	</li>
	<li>Add 100% formic acid to digested protein sample to decrease the pH to 2 - 3. 
	NOTE: This should require ~5 &#181;L per 1 mL of diluted peptide sample, but pH should be measured and more formic acid added if necessary.</li>
	<li>Load sample onto custom-made stage tip, centrifuge at 1000 x g for 1 min until all of sample goes through the tip.</li>
	<li>Wash bound peptides with 50 &#181;L 0.1% TFA. Centrifuge at 1000 x g for 1 min.</li>
	<li>Elute peptides by adding 50 &#181;L elution buffer (75% acetonitrile, 0.025% TFA) to stage tip and centrifuge at 1000 x g for 1 min to force the elution buffer out of the tip.</li>
	<li>Dry the sample using a vacuum centrifuge set at 150 x g and 1 - 3 kPa for 1 h. 
	NOTE: Dried peptides can be stored at -80 &#176;C indefinitely. This is a potential stopping point.</li>
</ol>
7. Removal of Crosslinked RNA
NOTE: Treat peptides with nuclease to remove crosslinked RNA.
<ol>
	<li>Resuspend the pellet in 50 &#181;L 2x nuclease buffer (100 mM NH4HCO3, 4 mM MgCl2).</li>
	<li>Measure peptide concentration using absorbance at 280 nm assuming 1 mg/mL of peptides for an A280 of 1. 
	NOTE: The expected concentration is 1 mg/mL.</li>
	<li>Adjust all samples to 1 mg/mL or to the lowest concentration using 2x nuclease buffer.</li>
	<li>Prepare a master mix of 50 &#181;L H2O + 1 &#181;L high purity nuclease (&#8805;250 U) (refer to the Table of Materials) per sample.</li>
	<li>Take 50 &#181;L of peptide sample and add 50 &#181;L of H2O + nuclease master mix.</li>
	<li>Incubate at 37 &#176;C for 1 h. Proceed to perform mass spectrometry. 
	NOTE: The peptides are now ready for mass spectrometry. They can be analyzed immediately or stored at -80 &#176;C indefinitely. This is a potential stopping point.</li>
</ol>
8. Nano Liquid Chromatography, Mass Spectrometry, and Raw Data Processing
NOTE: Because 4SU-crosslinking changes the mass of the peptide, their ions do not count toward the intensity of the non-crosslinked peptide during LC-MS/MS, which therefore appears to be decreased by the crosslinking. The degree and consistency of this decrease reflects the degree of protein-RNA crosslinking for each peptide24.
<ol>
	<li>Prepare HPLC buffers as follows: 0.1% formic acid in HPLC-grade water (buffer A); 0.1% formic acid in HPLC-grade acetonitrile (Buffer B).</li>
	<li>If a nano-HPLC is available, connect a nano-column with the following specifications: internal diameter 75 &#181;m; packed with C18-AQ particles; length 20 - 25 cm. 
	NOTE: If the HPLC available runs at higher flow rates use a proper column size for the indicated flow rate. Higher flow chromatography decreases sensitivity.</li>
	<li>Program the HPLC method as follows: flow rate 250 - 300 nL/min; gradient from 2 to 28% buffer B in 120 min, from 28 to 80% B for the next 5 min and isocratic 80% B for 10 min. 
	NOTE: If the HPLC does not have an automated column equilibration prior sample loading, start the program with 10 min at 0% B before loading the sample.</li>
	<li>Program the MS acquisition method to perform data-dependent acquisition (DDA) as standard shotgun proteomics experiments. The instrument duty cycle should alternate full MS scans with tandem MS scans (or MS/MS) of the most intense signals. Use the optimal settings for proteomics MS runs. 
	NOTE: For confident results, use instruments that can perform at least the full MS scan in high resolution mode (e.g. orbitrap, time-of-flight). For accurate quantification, make sure that intervals between full MS scans are no longer than 3 s. Longer duty cycles might prevent accurate definition of extracted ion chromatograms.</li>
	<li>Load 1 - 2 &#181;g of sample onto the HPLC column and run the HPLC-MS/MS method as programmed. For each biological replicate perform at least two independent runs and treat them as technical replicates.</li>
	<li>After MS, import the raw files into a proteomics software package for MS/MS spectra identification and peptide quantification via extracted ion chromatography. We recommend software packages that can perform chromatographic alignment of multiple MS runs (see the Table of Materials)29,30.</li>
	<li>If the option is available in the software suite, enable &#34;Match Between Runs&#34; prior to starting data processing.</li>
	<li>Once the analysis is finished, export the peptide list together with the quantification values.</li>
</ol>

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The authors have nothing to disclose.

We describe a detailed experimental protocol to perform RBR-ID in mESCs and, with appropriate modifications, in any cell that can incorporate 4SU into RNA. Other cell types may require optimization of the approach to ensure a sufficient signal to noise ratio. Additionally, while the protocol described herein focuses on the examination of nuclear RBPs, the RBR-ID technology should be easily adapted to different cellular compartments, such as the cytosol or specific organelles, by use of different fractionation strategies....

The purpose of the RBR-ID method is to identify novel RNA-binding proteins (RBPs) and map their RNA-binding regions (RBRs) with peptide-level resolution to facilitate the design of RNA-binding mutants and the investigation of the biological and biochemical functions of protein-RNA interactions.
RNA is unique among biomolecules as it can both act as a messenger carrying genetic information and also fold into complex three-dimensional structures with biochemical functions more akin to those of proteins1,2. A growing body of evidence suggests that noncoding RNAs (ncRNAs) play important roles in various gene regulatory and epigenetic pathways3,4,5 and, typically, these regulatory functions are mediated in concert with proteins that interact specifically with a given RNA. Of particular relevance, a set of interacting proteins was recently identified for the intensely studied long ncRNA (lncRNA) Xist, providing valuable insight into how this lncRNA mediates X-chromosome inactivation in female cells6,7,8. Notably, several of these Xist-interacting proteins do not contain any canonical RNA-binding domains9, and therefore their RNA-binding activity could not be predicted in silico based on their primary sequence alone. Considering that thousands of lncRNAs are expressed in any given cell10, it is reasonable to assume that many of them might act via interactions with yet to be discovered RNA-binding proteins (RBPs). An experimental strategy to identify these novel RBPs would therefore greatly facilitate the task of dissecting the biological function of ncRNAs.
Previous attempts to identify RBPs empirically have relied on polyA+ RNA selection coupled to mass spectrometry (MS)11,12,13,14,15. Although these experiments added many proteins to the list of putative RBPs, by design they could only detect proteins bound to polyadenylated transcripts. However, most small RNAs and many lncRNAs are not polyadenylated.16,17 and their interacting proteins would likely have been missed in these experiments. A recent study applied machine learning to protein-protein interactome databases to identify proteins that co-purified with multiple known RBPs and showed that these recurrent RBP partners were more likely to possess RNA-binding activities18. However, this approach relies on mining existing large interaction databases and can only identify proteins that can be co-purified in non-denaturing conditions with known RBPs, thus excluding from the analysis insoluble, membrane-embedded, and scarce proteins.
The identification of a protein as a bona fide RBP often does not automatically yield information on the biological and/or biochemical function of the protein-RNA interaction. To address this point, it is typically desirable to identify the protein domain and amino acid residues involved in the interaction so that specific mutants can be designed to test the function of RNA binding in the context of each novel RBP19,20. Previous efforts by our group and others have used recombinant protein fragments and deletion mutants to identify RNA binding regions (RBRs)19,20,21,22; however, such approaches are labor-intensive and incompatible with high-throughput analyses. More recently, a study described an experimental strategy to map RNA-binding activities in a high-throughput fashion using mass spectrometry23; however, this approach relied on a double polyA+ RNA selection, and thus carried the same limitations as the RBP identification approaches described above.
We developed a technique, termed RNA binding region identification (RBR-ID), which exploits protein-RNA photocrosslinking and quantitative mass spectrometry to identify proteins and protein regions interacting with RNA in live cells without making assumption on the RNA polyadenylation status, thus including RBPs bound to polyA- RNAs24. Moreover, this method relies exclusively on crosslinking and has no requirements on protein solubility or accessibility and is thus suitable to map RNA-binding activities within membranes (e.g. the nuclear envelope) or poorly soluble compartments (e.g. the nuclear matrix). We describe the experimental steps to perform RBR-ID for the nuclei of mouse embryonic stem cells (mESCs) but with minor modifications this protocol should be suitable for a variety of cell types, provided that they can efficiently incorporate 4SU from the culture medium.

<table>
	<tbody>
		<tr>
			<td>KnockOut DMEM</td>
			<td>Fisher Scientific</td>
			<td>10829018</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum, qualified, US origin</td>
			<td>Fisher Scientific</td>
			<td>26140079</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine solution 200 mM</td>
			<td>Sigma</td>
			<td>G7513</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin solution</td>
			<td>Sigma</td>
			<td>P0781</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM Non-essential Amino Acid Solution (100&#215;)</td>
			<td>Sigma</td>
			<td>M7145</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Sigma</td>
			<td>M3148</td>
			<td></td>
		</tr>
		<tr>
			<td>ESGRO Leukemia Inhibitory Factor (LIF)</td>
			<td>EMD Millipore</td>
			<td>ESG1106</td>
			<td></td>
		</tr>
		<tr>
			<td>CHIR99021</td>
			<td>Tocris</td>
			<td>4423</td>
			<td></td>
		</tr>
		<tr>
			<td>PD0325901</td>
			<td>Sigma</td>
			<td>PZ0162</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelatin solution,2% in water</td>
			<td>Sigma</td>
			<td>G1393</td>
			<td></td>
		</tr>
		<tr>
			<td>4-thiouridine</td>
			<td>Sigma</td>
			<td>T4509</td>
			<td>50 mM stock in water</td>
		</tr>
		<tr>
			<td>Spectrolinker XL-1500</td>
			<td>Fisher Scientific</td>
			<td>11-992-90</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenylmethanesulfonyl fluoride</td>
			<td>Sigma</td>
			<td>78830</td>
			<td></td>
		</tr>
		<tr>
			<td>IGEPAL CO-630</td>
			<td>Sigma</td>
			<td>542334</td>
			<td>Commercial form of octyl-phenoxy-polyethoxy-ethanol detergent</td>
		</tr>
		<tr>
			<td>Iodoacetamide</td>
			<td>Sigma</td>
			<td>I6125</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin, sequencing grade</td>
			<td>Promega</td>
			<td>V5111</td>
			<td></td>
		</tr>
		<tr>
			<td>Empore solid phase extraction disk</td>
			<td>3M</td>
			<td>66883</td>
			<td></td>
		</tr>
		<tr>
			<td>OLIGO R3 Reversed - Phase Resin</td>
			<td>Fisher Scientific</td>
			<td>1133903</td>
			<td></td>
		</tr>
		<tr>
			<td>Benzonase</td>
			<td>Sigma</td>
			<td>E8263</td>
			<td>High purity nuclease</td>
		</tr>
		<tr>
			<td>Sonic Dismembrator Model 100</td>
			<td>Fisher Scientific</td>
			<td>discontinued</td>
			<td>updated with FB505110</td>
		</tr>
		<tr>
			<td>HPLC grade acetonitrile</td>
			<td>Fisher Chemical</td>
			<td>A955-4</td>
			<td></td>
		</tr>
		<tr>
			<td>HPLC grade water</td>
			<td>Fisher Scientific</td>
			<td>W6 4</td>
			<td></td>
		</tr>
		<tr>
			<td>TFA</td>
			<td>Fisher Scientific</td>
			<td>A11650</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium Bicarbonate</td>
			<td>Sigma</td>
			<td>A6141</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic Acid</td>
			<td>Sigma</td>
			<td>49199</td>
			<td></td>
		</tr>
		<tr>
			<td>Formic Acid</td>
			<td>Sigma</td>
			<td>F0507</td>
			<td></td>
		</tr>
		<tr>
			<td>ReproSil-Pur 18-AQ</td>
			<td>Dr. Maisch GmbH HPLC</td>
			<td>r13.aq.0003</td>
			<td>Packing material for HPLC column</td>
		</tr>
		<tr>
			<td>Capillary for nano columns (75 &#181;m)</td>
			<td>Molex</td>
			<td>1068150017</td>
			<td></td>
		</tr>
		<tr>
			<td>MaxQuant software</td>
			<td>Max Planck Institute for Biochemistry</td>
			<td></td>
			<td>Can perform chromatographic alignment of multiple MS runs</td>
		</tr>
	</tbody>
</table>

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.

Figure 1 depicts the RBR-ID workflow. Due to the relatively low crosslinking efficiency of this technique, it is very important to consider both the depletion level and consistency of the observed effect (P-value) across biological replicates. Figure 2 shows a volcano plot of RBR-ID result. Peptides that overlapped RNA recognition motif (RRM) domain show highly consistent depletion level. RRM domains can be used as a pos...

Watch this Scientific Journal Video about Sample Preparation for Mass Spectrometry-based Identification of RNA-binding Regions at JoVE.com

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

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 ...

sample-preparation-for-mass-spectrometry-based-identification-rna

Research

JoVE Journal

Biochemistry

8.0K Views.  University of Pennsylvania Perelman School of Medicine. 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.

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

A Tandem Liquid Chromatography&#8211;Mass Spectrometry-based Approach for Metabolite Analysis of Staphylococcus aureus

In an effort to thwart bacterial pathogens, hosts often limit the availability of nutrients at the site of infection. This limitation can alter the abundances of key metabolites to which regulatory factors respond, adjusting cellular metabolism. In recent years, a number of proteins and RNA have emerged as important regulators of virulence gene expression. For example, the CodY protein responds to levels of branched-chain amino acids and GTP and is widely conserved in low G+C Gram-positive bacteria. As a global regulator in Staphylococcus aureus, CodY controls the expression of dozens of virulence and metabolic genes. We hypothesize that S. aureus uses CodY, in part, to alter its metabolic state in an effort to adapt to nutrient-limiting conditions potentially encountered in the host environment. This manuscript describes a method for extracting and analyzing metabolites from S. aureus using liquid chromatography coupled with mass spectrometry, a protocol that was developed to test this hypothesis. The method also highlights best practices that will ensure rigor and reproducibility, such as maintaining biological steady state and constant aeration without the use of continuous chemostat cultures. Relative to the USA200 methicillin-susceptible S. aureus isolate UAMS-1 parental strain, the isogenic codY mutant exhibited significant increases in amino acids derived from aspartate (e.g., threonine and isoleucine) and decreases in their precursors (e.g., aspartate and O-acetylhomoserine). These findings correlate well with transcriptional data obtained with RNA-seq analysis: genes in these pathways were up-regulated between 10- and 800-fold in the codY null mutant. Coupling global analyses of the transcriptome and the metabolome can reveal how bacteria alter their metabolism when faced with environmental or nutritional stress, providing potential insight into the physiological changes associated with nutrient depletion experienced during infection. Such discoveries may pave the way for the development of novel anti-infectives and therapeutics.

Here we describe a protocol for the extraction of metabolites from Staphylococcus aureus and their subsequent analysis via liquid chromatography and mass spectrometry.

In an effort to thwart bacterial pathogens, hosts often limit the availability of nutrients at the site of infection. This limitation can alter the ...

Capture and Identification of RNA-binding Proteins by Using Click Chemistry-assisted RNA-interactome Capture (CARIC) Strategy

A comprehensive identification of RNA-binding proteins (RBPs) is key to understanding the posttranscriptional regulatory network in cells. A widely used strategy for RBP capture exploits the polyadenylation [poly(A)] of target RNAs, which mostly occurs on eukaryotic mature mRNAs, leaving most binding proteins of non-poly(A) RNAs unidentified. Here we describe the detailed procedures of a recently reported method termed click chemistry-assisted RNA-interactome capture (CARIC), which enables the transcriptome-wide capture of both poly(A) and non-poly(A) RBPs by combining the metabolic labeling of RNAs, in vivo UV cross-linking, and bioorthogonal tagging.

Capture and Identification of RNA-binding Proteins by Using Click Chemistry-assisted RNA-interactome Capture CARIC Strategy

A detailed protocol for applying the click chemistry-assisted RNA-interactome capture (CARIC) strategy to identify proteins binding to both coding and noncoding RNAs is presented.

A comprehensive identification of RNA-binding proteins (RBPs) is key to understanding the posttranscriptional regulatory network in cells. A widely used ...

Identification of Functional Protein Regions Through Chimeric Protein Construction

The goal of this protocol encompasses the design of chimeric proteins in which distinct regions of a protein are replaced by their corresponding sequences in a structurally similar protein, in order to determine the functional importance of these regions. Such chimeras are generated by means of a nested PCR protocol using overlapping DNA fragments and adequately designed primers, followed by their expression within a mammalian system to ensure native secondary structure and post-translational modifications. 
The functional role of a distinct region is then indicated by a loss of activity of the chimera in an appropriate readout assay. In consequence, regions harboring a set of critical amino acids are identified, which can be further screened by complementary techniques (e.g. site-directed mutagenesis) to increase molecular resolution. Although limited to cases in which a structurally related protein with differing functions can be found, chimeric proteins have been successfully employed to identify critical binding regions in proteins such as cytokines and cytokine receptors. This method is particularly suitable in cases in which the protein's functional regions are not well defined, and constitutes a valuable first step in directed evolution approaches to narrow down the regions of interest and reduce the screening effort involved.

Structurally related proteins frequently exert distinct biological functions. The exchange of equivalent regions of these proteins in order to create chimeric proteins constitutes an innovative approach to identify critical protein regions that are responsible for their functional divergence.

The goal of this protocol encompasses the design of chimeric proteins in which distinct regions of a protein are replaced by their corresponding sequences ...

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 ...

A Plasma Sample Preparation for Mass Spectrometry using an Automated Workstation

Sample preparation for mass spectrometry analysis in proteomics requires enzymatic cleavage of proteins into a peptide mixture. This process involves numerous incubation and liquid transfer steps in order to achieve denaturation, reduction, alkylation, and cleavage. Adapting this workflow onto an automated workstation can increase efficiency and reduce coefficients of variance, thereby providing more reliable data for statistical comparisons between sample types. We previously described an automated proteomic sample preparation workflow1. Here, we report the development of a more efficient and better controlled workflow with the following advantages: 1) The number of liquid transfer steps is reduced from nine to six by combining reagents; 2) Pipetting time is reduced by selective tip pipetting using a 96-position pipetting head with multiple channels; 3) Potential throughput is increased by the availability of up to 45 deck positions; 4) Complete enclosure of the system provides improved temperature and environmental control and reduces the potential for contamination of samples or reagents; and 5) The addition of stable isotope labeled peptides, as well as &#946;-galactosidase protein, to each sample makes monitoring and quality control possible throughout the entire process. These hardware and process improvements provide good reproducibility and improve intra-assay and inter-assay precision (CV of less than 20%) for LC-MS based protein and peptide quantification. The entire workflow for digesting 96 samples in a 96-well plate can be completed in approximately 5 hours.

Here, we present an automated plasma protein digestion method for mass spectrometry-based quantitative proteomic analysis. In this protocol, the liquid transferring and incubation steps for protein denaturation, reduction, alkylation, and trypsin digestion reactions are streamlined and automated. It takes approximately five hours to prepare a 96-well plate with desired precision.

Sample preparation for mass spectrometry analysis in proteomics requires enzymatic cleavage of proteins into a peptide mixture. This process involves ...

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 ...

Untargeted Liquid Chromatography-Mass Spectrometry-Based Metabolomics Analysis of Wheat Grain

Understanding the interactions between genes, the environment and management in agricultural practice could allow more accurate prediction and management of product yield and quality. Metabolomics data provides a read-out of these interactions at a given moment in time and is informative of an organism&#39;s biochemical status. Further, individual metabolites or panels of metabolites can be used as precise biomarkers for yield and quality prediction and management. The plant metabolome is predicted to contain thousands of small molecules with varied physicochemical properties that provide an opportunity for a biochemical insight into physiological traits and biomarker discovery. To exploit this, a key aim for metabolomics researchers is to capture as much of the physicochemical diversity as possible within a single analysis. Here we present a liquid chromatography-mass spectrometry-based untargeted metabolomics method for the analysis of field-grown wheat grain. The method uses the liquid chromatograph quaternary solvent manager to introduce a third mobile phase and combines a traditional reversed-phase gradient with a lipid-amenable gradient. Grain preparation, metabolite extraction, instrumental analysis and data processing workflows are described in detail. Good mass accuracy and signal reproducibility were observed, and the method yielded approximately 500 biologically relevant features per ionization mode. Further, significantly different metabolite and lipid feature signals between wheat varieties were determined.

A method for the untargeted analysis of wheat grain metabolites and lipids is presented. The protocol includes an acetonitrile metabolite extraction method and reversed phase liquid chromatography-mass spectrometry methodology, with acquisition in positive and negative electrospray ionization modes.

Understanding the interactions between genes, the environment and management in agricultural practice could allow more accurate prediction and management ...

TMT Sample Preparation for Proteomics Facility Submission and Subsequent Data Analysis

Proteomic technologies are powerful methodologies that can aid our understanding of mechanisms of action in biological systems by providing a global view of the impact of a disease, treatment, or other condition on the proteome as a whole. This report provides a detailed protocol for the extraction, quantification, precipitation, digestion, labeling, and subsequent data analysis of protein samples. Our optimized TMT labeling protocol requires a lower tag-label concentration and achieves consistently reliable data. We have used this protocol to evaluate protein expression profiles in a variety of mouse tissues (i.e., heart, skeletal muscle, and brain) as well as cells cultured in vitro. In addition, we demonstrate how to evaluate thousands of proteins from the resulting dataset.

We present an optimized tandem mass tag (TMT) labeling protocol that includes detailed information for each of the following steps: protein extraction, quantification, precipitation, digestion, labeling, submission to a proteomics facility, and data analyses.

Proteomic technologies are powerful methodologies that can aid our understanding of mechanisms of action in biological systems by providing a global view ...

A Mass Spectrometry-Based Proteomics Approach for Global and High-Confidence Protein R-Methylation Analysis

Protein Arginine (R)-methylation is a widespread protein post-translational modification (PTM) involved in the regulation of several cellular pathways, including RNA processing, signal transduction, DNA damage response, miRNA biogenesis, and translation.
In recent years, thanks to biochemical and analytical developments, mass spectrometry (MS)-based proteomics has emerged as the most effective strategy to characterize the cellular methyl-proteome with single-site resolution. However, identifying and profiling in vivo protein R-methylation by MS remains challenging and error-prone, mainly due to the substoichiometric nature of this modification and the presence of various amino acid substitutions and chemical methyl-esterification of acidic residues that are isobaric to methylation. Thus, enrichment methods to enhance the identification of R-methyl-peptides and orthogonal validation strategies to reduce False Discovery Rates (FDR) in methyl-proteomics studies are required.
Here, a protocol specifically designed for high-confidence R-methyl-peptides identification and quantitation from cellular samples is described, which couples metabolic labeling of cells with heavy isotope-encoded Methionine (hmSILAC) and dual protease in-solution digestion of whole cell extract, followed by off-line High-pH Reversed Phase (HpH-RP) chromatography fractionation and affinity enrichment of R-methyl-peptides using anti-pan-R-methyl antibodies. Upon high-resolution MS analysis, raw data are first processed with the MaxQuant software package and the results are then analyzed by hmSEEKER, a software designed for the in-depth search of MS peak pairs corresponding to light and heavy methyl-peptide within the MaxQuant output files.

Protein Arginine (R)-methylation is a wide-spread post-translational modification regulating multiple biological pathways. Mass spectrometry is the best technology to globally profile the R-methyl-proteome, when coupled to biochemical approaches for modified peptide enrichment. The workflow designed for the high confidence identification of global R-methylation in human cells is described here.

Protein Arginine (R)-methylation is a widespread protein post-translational modification (PTM) involved in the regulation of several cellular pathways, ...

Preparation of High-Temperature Sample Grids for Cryo-EM

The sample grids for cryo-electron microscopy (cryo-EM) experiments are usually prepared at a temperature optimal for the storage of biological samples, mostly at 4 &#176;C and occasionally at room temperature. Recently, we discovered that the protein structure solved at low temperature may not be functionally relevant, particularly for proteins from thermophilic archaea. A procedure was developed to prepare protein samples at higher temperatures (up to 70 &#176;C) for cryo-EM analysis. We showed that the structures from samples prepared at higher temperatures are functionally relevant and temperature dependent. Here we describe a detailed protocol for preparing sample grids at high temperature, using 55 &#176;C as an example. The experiment made use of a vitrification apparatus modified using an additional centrifuge tube, and samples were incubated at 55 &#176;C. The detailed procedures were fine-tuned to minimize vapor condensation and obtain a thin layer of ice on the grid. Examples of successful and unsuccessful experiments are provided.

This paper provides a detailed protocol for preparing sample grids at temperatures as high as 70 &#176;C, prior to plunge freezing for cryo-EM experiments.

The sample grids for cryo-electron microscopy (cryo-EM) experiments are usually prepared at a temperature optimal for the storage of biological samples, ...