This work was funded in part by an NIH Pathway to Independence Award (grant GM 099893) and faculty startup funds to SRB, as well as a Research Project Grant (grant GM 042219). The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

1. Preparation of Buffer Solutions
<ol>
	<li>Prepare phosphate-buffered saline (PBS; pH 7.4) by diluting a stock solution of 10x PBS to a final concentration of 1x with ultrapure (distilled and deionized) water.</li>
	<li>Prepare quenching solution by combining 2 mL of acetonitrile, 2 mL of methanol, 1 mL of ultrapure H2O, and 19 &#181;L (0.1 mM final concentration) of formic acid.</li>
	<li>Prepare LC-MS solvent A by adding formic acid (0.2% [v/v] final concentration) to ultrapure water.</li>
	<li>Prepare LC-MS solvent B by adding formic acid (0.2% [v/v] final concentration) to acetonitrile. 
	NOTE: All solutions should be prepared using the highest-purity reagents available (generally high-performance liquid chromatography grade). Solutions should be prepared fresh before each experiment and stored on ice prior to use.</li>
</ol>
2. Establishment of Steady-state S. aureus Growth
<ol>
	<li>Streak S. aureus strains of interest for isolation on tryptic soy agar (TSA) from a frozen glycerol stock. Incubate at 37 &#176;C for 16-24 h.</li>
	<li>Inoculate 4 mL of tryptic soy broth (TSB) or another suitable medium in sterile glass incubation tubes with single colonies of each strain. Incubate inclined (~70&#176; angle) with rotation at 60 rotations per min (rpm) at 37 &#176;C for 16-20 h. 
	NOTE: Overnight cultures are prone to oxygen gradients when using standard methods, including those described in step 2.2, which affect cellular physiology. Thus, we employ a multiple back-dilution strategy to ensure biological steady state (see steps 2.4-3.2, below).</li>
	<li>Use a spectrophotometer to measure the optical density of the cultures from step 2.2 at 600 nm (OD600). Use sterile medium as an optical reference (blank). Dilute these cells to an OD600 of 0.05 in 50 mL of sterile TSB medium (pre-warmed to 37 &#176;C) in separate, 250 mL DeLong flasks.</li>
	<li>Incubate the cultures at 37 &#176;C in a water bath with shaking at 280 rpm.</li>
	<li>Every 30 min, take OD600 measurements; as the optical densities increase, it may become necessary to dilute the cultures with TSB so that they remain within the linear absorbance range of the spectrophotometer.</li>
	<li>When cultures from step 2.5 achieve an OD600 of ~0.8-1.0, subculture them into 50 mL of 37 &#176;C TSB to an OD600 of 0.01-0.05 and repeat steps 2.4 and 2.5.</li>
</ol>
3. Sample Collection Setup
<ol>
	<li>Prepare a bed of crushed dry ice in an appropriate vessel (e.g., glass dish, ice bucket, or cooler).</li>
	<li>As the optical densities of the cultures approach the desired harvest point, add 1 mL of quenching solution to a 35 mm untreated Petri dish and pre-cool on dry ice for &#8805;5 min. 
	NOTE: The &#34;desired harvest point&#34; will vary depending on experimental goals. For example, if one were to examine metabolites during aerobic growth, key indicators of this state include acetate excretion and re-assimilation of the acetate during the post-exponential growth phase32,33. Generally, this point should be within a specific growth stage (e.g., exponential phase). The specific OD600 values associated with this stage may vary between different bacterial strains and growth media.</li>
	<li>Place a stainless steel filter frit (pre-chilled to -20 &#176;C) in a rubber stopper and place it atop a vacuum flask attached to a house vacuum or vacuum pump.</li>
	<li>Apply the vacuum and place a mixed cellulose ester membrane (0.22 &#181;m pore size) on top. 
	NOTE: It is critical to use a filter with a diameter equal to that of the frit and to properly center this filter to ensure that the sample is drawn through the filter rather than over the edge. Wetting the membrane with ice-cold, sterile H2O may help with positioning the membrane.</li>
</ol>
4. Sample Harvest
<ol>
	<li>At an OD600 of ~0.4-0.5, use a serological pipette to remove 13 mL of culture from the flask and to apply the sample to the filter.</li>
	<li>After the entire sample has been filtered, immediately wash the filter with &#8805;5 mL of ice-cold PBS to wash away medium-associated metabolites.</li>
	<li>Disconnect the vacuum and use a pair of sterile tweezers to remove the filter from the frit. Invert the filter (cell-side down) into the pre-chilled quench solution. 
	NOTE: It is important to perform the above steps quickly (i.e., within seconds) and as soon as the liquid has been removed to ensure the rapid quenching of the cells, arresting metabolic activity.</li>
	<li>Incubate the filter in quench solution on dry ice for &#8805;20 min.</li>
	<li>Using sterile tweezers, invert the filter (cell-side up) in the petri dish and use a micropipette to rinse the cells off of the membrane into the quench solution.</li>
	<li>Re-suspended the cells in quench solution and then transfer the cell suspension to a sterile 2 mL impact-resistant tube containing ~100 &#181;L of 0.1 mm silica beads. Store this on dry ice or at -80 &#176;C.</li>
</ol>
5. Metabolite Extraction
<ol>
	<li>Thaw samples on wet ice and disrupt the cells in a homogenizer with four 30 s bursts at 6,000 rpm, with 2 min cooling periods on dry ice between cycles.</li>
	<li>Clarify the lysates for 15 min in a pre-chilled, refrigerated microcentrifuge at maximum speed (i.e., 18,213 x g at &#8804;4 &#176;C).</li>
	<li>Transfer the supernatant to a clean microcentrifuge tube.</li>
	<li>Using a micropipette, transfer a small portion of the sample to a microcentrifuge tube for the quantification of residual peptide content in step 6; store the remainder at -80 &#176;C. 
	NOTE: The volume of reserved sample varies, depending on the BCA assay used in step 6.1. This sample should be stored on wet ice for immediate analysis or frozen at -80 &#176;C.</li>
</ol>
6. Bicinchoninic Acid (BCA) Assay
<ol>
	<li>Perform a BCA assay as recommended by the kit manufacturer, using samples from step 5.4 to determine the residual peptide concentration for each sample.</li>
</ol>
7. LC-MS
<ol>
	<li>Mix 75 &#181;L of S. aureus extract with 75 &#181;L of LC-MS solvent B, prepared in step 1.4.</li>
	<li>Vortex to mix and spin at 13,000 x g for 5 min.</li>
	<li>Place 100 &#181;L of supernatant into a liquid chromatography (LC) vial and cap it. Ensure that no air bubbles are trapped in the sample.</li>
	<li>Load the LC vials onto the LC-MS autosampler and edit the running list in the software &#34;Offline Worklist Editor.&#34;
	<ol>
		<li>Fill out the &#34;Sample Name&#34; (e.g., wild-type-1), &#34;Sample Position&#34; (e.g., P1-A1), &#34;Method&#34; (e.g., Formic Acid-Negative Method), and &#34;Data File&#34; (e.g., wild-type-1) columns. Click the button &#34;Save Worklist&#34; button. Open the &#34;Mass Spectrometry Data Acquisition Workstation&#34; software and input the previously saved worklist. Click the &#34;Start Worklist Run&#34; button to start the continuous LC-MS measurement.</li>
	</ol>
	</li>
	<li>Separate the samples on a column, link the column to a time of flight (TOF) spectrometer, and couple the TOF spectrometer with the LC system. Use a mobile-phase gradient as follows: 0-2 min, 85% solvent B; 3-5 min, 80% solvent B; 6-7 min, 75% solvent B; 8-9 min, 70% solvent B; 10-11.1 min, 50% solvent B; 11.1-14 min, 20% solvent B; and 14.1-24 min, 5% solvent B; end with a 10 min re-equilibration period at 85% solvent B and a flow rate of 0.4 mL min-1.</li>
	<li>Using an isocratic pump, infuse a reference mass solution with the run to allow for simultaneous mass axis calibration. 
	NOTE: This step is based on the standard TOF spectrometer manual.
	<ol>
		<li>Use the mixture of acetic acid D4 and hexakis (1H,1H,3H-tetrafluoropropoxy) phosphazine as the reference mass solution to perform the real-time calibration. Use the isocratic pump with the flow rate of 2.5 mL min-1 for the infusion.</li>
	</ol>
	</li>
</ol>
8. Batch Correction of Ion Counts
<ol>
	<li>Designate any sample to serve as a reference sample for batch correction (e.g., wild-type, replicate 1).</li>
	<li>Calculate the sum of the ion counts for all metabolites within the reference sample. Repeat this calculation for all samples.</li>
	<li>Divide the total ion count of each sample by the total ion count of the reference sample to generate a ratio.</li>
	<li>Divide the ion count for each metabolite within a sample by the sample/reference ratio to obtain a batch-corrected ion count for each metabolite.</li>
</ol>
9. Peptide Normalization
<ol>
	<li>Divide the batch-corrected ion count values for each sample obtained in step 8 by the peptide concentration determined with the BCA assay in step 6 to yield a normalized value for each metabolite. 
	NOTE: The normalized, batch-batch corrected ion counts for each metabolite obtained in step 9.1 can be directly compared between strains and subjected to statistical analysis (e.g., a Mann-Whitney U-test). Alternatively, a metabolite known to be unchanged either by the treatment or genetic background may be used as a normalizer to detect changes due to metabolite decomposition. The inclusion of a known amount of L-norvaline or gluraric acid in the extraction buffer can be used to correct for loss during sample processing34.</li>
</ol>

The authors declare that they have no competing financial interests.

All small-molecule metabolites are connected to one another through their common origins in central metabolic pathways. During exponential growth, bacterial cells are at biological and metabolic steady state, providing a snapshot of the physiological state under specific conditions. CodY monitors nutrient sufficiency by responding to ILV and GTP. As ILV and GTP pools drop, CodY activity is likely progressively reduced, adjusting the expression of its target genes to adapt to increasing nutrient depletion<sup class="xref"...

Bacterial pathogens must contend with many challenges within the host environment. In addition to direct attack by immune cells, the host also sequesters nutrients essential for bacterial survival and replication, generating nutritional immunity1,2. To survive these hostile environments, bacterial pathogens deploy virulence factors. Some of these factors allow the bacteria to evade the immune response; other factors include secreted digestive enzymes, such as hyaluronidase, thermonuclease, and lipase, which may enable the bacteria to replenish missing nutrients by consuming tissue-derived constituents3,4,5. Indeed, bacteria have evolved regulatory systems that tie the physiological state of the cell to the production of virulence factors6,7,8,9,10.
A growing body of evidence points to CodY as a critical regulator linking metabolism and virulence. Although first discovered in Bacillus subtilis as a repressor of the dipeptide permease (dpp) gene11, CodY is now known to be produced by nearly all the low G+C Gram-positive bacteria12,13 and regulates dozens of genes involved in carbon and nitrogen metabolism14,15,16,17,18,19. In pathogenic species, CodY also controls the expression of some of the most important virulence genes20,21,22,23,24,25,26,27. CodY is activated as a DNA-binding protein by two classes of ligands: branched-chain amino acids (BCAAs; isoleucine, leucine, and valine [ILV]) and GTP. When these nutrients are abundant, CodY represses (or in some cases, stimulates) transcription. As these nutrients become limited, CodY activity is progressively reduced, resulting in a graded transcriptional response that re-routes precursors through various metabolic pathways connected to central metabolism28,29,30. 
Tandem liquid chromatography coupled to mass spectrometry (LC-MS) is a powerful technique that can accurately identify and quantify small-molecule intracellular metabolites31. When paired with transcriptome analysis (e.g., RNA-Seq), this analytical workflow can provide insight into the physiological changes that occur in response to environmental or nutritional stress. Here, we present a method for metabolite extraction from Staphylococcus aureus cells and subsequent analysis via LC-MS. This approach has been used to demonstrate the pleiotropic effects of CodY on S. aureus physiology.

<table border="0" cellpadding="0" cellspacing="0" width="933">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">Material/Equipmenta</td>
			<td style="width:167px;"></td>
			<td style="width:163px;"></td>
			<td style="width:241px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:363px;">DeLong Culture Flask (250 ml)</td>
			<td style="width:167px;">Belco</td>
			<td>2510-00250</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:363px;">Sidearm Flask, 500 ml</td>
			<td style="width:167px;">Pyrex</td>
			<td style="width:163px;">5340</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:363px;">3-hole Rubber Stopper, #7</td>
			<td style="width:167px;">Fisher</td>
			<td style="width:163px;">14-131E</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:363px;">Stainless Steel Filter holder/frit</td>
			<td style="width:167px;">VWR</td>
			<td>89428-936</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:363px;">Petri Dish, 35 mm</td>
			<td style="width:167px;">Corning</td>
			<td style="width:163px;">430588</td>
			<td>Not tissue culture treated</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:363px;">Mixed cellulose ester membrane, 0.22 &#956;m pore size</td>
			<td style="width:167px;">Millipore</td>
			<td>GSWP02500</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:363px;">Impact-resistant tubes, 2 ml</td>
			<td style="width:167px;">USA Scientific</td>
			<td style="width:163px;">1420-9600</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:363px;">Silica Beads, 0.1 mm</td>
			<td style="width:167px;">Biospec Products Inc</td>
			<td>11079101Z</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:363px;">Precellys 24 homogenizer</td>
			<td style="width:167px;">Bertin Instruments</td>
			<td style="width:163px;">EQ03119-200-RD000.0</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:363px;">Micro BCA Protein Assay Kit</td>
			<td style="width:167px;">Pierce (Thermo Scientific)</td>
			<td style="width:163px;">23235</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:363px;">Cogent Diamond hydride type C column</td>
			<td style="width:167px;">Agilent</td>
			<td style="width:163px;">70000-15P-2</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:363px;">Accurate-Mass Time-of-Flight (TOF) LC-MS, 6200 Series</td>
			<td style="width:167px;">Agilent</td>
			<td style="width:163px;">G6230B</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:363px;">Quat Pump, 1290 Series</td>
			<td style="width:167px;">Agilent</td>
			<td style="width:163px;">G4204A&#160;</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:363px;">Bin Pump, 1290 Series</td>
			<td style="width:167px;">Agilent</td>
			<td style="width:163px;">G4220A&#160;</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:363px;">Valve Drive, 1290 Series</td>
			<td style="width:167px;">Agilent</td>
			<td style="width:163px;">G1107A&#160;</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:363px;">Isocratic Pump, 1290 Series</td>
			<td style="width:167px;">Agilent</td>
			<td style="width:163px;">G1310B&#160;</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:363px;">TCC, 1290 Series</td>
			<td style="width:167px;">Agilent</td>
			<td style="width:163px;">G1316C&#160;</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:363px;">Sampler, 1290 Series</td>
			<td style="width:167px;">Agilent</td>
			<td style="width:163px;">G4226A&#160;</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:363px;">Thermostat, 1290 Series</td>
			<td style="width:167px;">Agilent</td>
			<td style="width:163px;">G1330B&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Name</td>
			<td style="width:167px;">Company</td>
			<td style="width:163px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:363px;">Chemical</td>
			<td style="width:167px;"></td>
			<td style="width:163px;"></td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:363px;">Tryptic Soy Broth</td>
			<td style="width:167px;">Becton Dickinson</td>
			<td style="width:163px;">211825</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:363px;">Difco Agar, Granulated</td>
			<td style="width:167px;">Becton Dickinson</td>
			<td style="width:163px;">214530</td>
			<td>Solid media contains 1.5% [w/v] agar</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:363px;">Phosphate-buffered saline (pH 7.4) 10X</td>
			<td style="width:167px;">Ambion</td>
			<td style="width:163px;">AM9624</td>
			<td>Dilute fresh to 1X with ultra-pure water</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:363px;">Acetonitrile</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td style="width:163px;">A955-500</td>
			<td>Optima LC-MS</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:363px;">Methanol</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td style="width:163px;">A456-500</td>
			<td>Optima LC-MS; toxic</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:363px;">Formic Acid</td>
			<td style="width:167px;">Sigma Aldrich</td>
			<td style="width:163px;">94318</td>
			<td>For mass spectrometry, 98%</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Name</td>
			<td style="width:167px;">Company</td>
			<td style="width:163px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:363px;">Software</td>
			<td style="width:167px;"></td>
			<td style="width:163px;"></td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:363px;">MassHunter</td>
			<td style="width:167px;">Agilent</td>
			<td style="width:163px;">G3337AA</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:363px;">Bacterial Strain</td>
			<td style="width:167px;">Species</td>
			<td style="width:163px;">Strain</td>
			<td>Genotype</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:363px;">SRB 337</td>
			<td style="width:167px;">Staphylococcus aureus</td>
			<td style="width:163px;">USA200 MSSA UAMS-1</td>
			<td>wild type</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:363px;">SRB 372</td>
			<td style="width:167px;">Staphylococcus aureus</td>
			<td style="width:163px;">USA200 MSSA UAMS-1</td>
			<td>&#916;codY::erm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:933px;">aChemicals and materials listed are specific to the method described and do not include standard laboratory chemicals or supplies.</td>
		</tr>
	</tbody>
</table>

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.

We have analyzed intracellular metabolite pools in S. aureus during in vitro growth in a rich, complex medium. As proof of principle, we compared metabolite profiles between the methicillin-susceptible S. aureus osteomyelitis isolate UAMS-1 (wild-type [WT]) and an isogenic strain lacking the global transcriptional regulator CodY (&#916;codY)26. Steady-state, exponential cultures of the WT and codY strains were establishe...

Watch this Scientific Journal Video about A Tandem Liquid ChromatographyÃ¢â‚¬â€œMass Spectrometry-based Approach for Metabolite Analysis of Staphylococcus aureus at JoVE.com

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

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 ...

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10.0K Views.  Georgetown University. Here we describe a protocol for the extraction of metabolites from Staphylococcus aureus and their subsequent analysis via liquid chromatography and mass spectrometry.

Video: A Tandem Liquid Chromatography–Mass Spectrometry-based Approach for Metabolite Analysis of Staphylococcus aureus

A Facile and Efficient Approach for the Production of Reversible Disulfide Cross-linked Micelles

Nanomedicine is an emerging form of therapy that harnesses the unique properties of particles that are nanometers in scale for biomedical application. Improving drug delivery to maximize therapeutic outcomes and to reduce drug-associated side effects are some of the cornerstones of present-day nanomedicine. Nanoparticles in particular have found a wide application in cancer treatment. Nanoparticles that offer a high degree of flexibility in design, application, and production based on the tumor microenvironment are projected to be more effective with rapid translation into clinical practice. The polymeric micellar nano-carrier is a popular choice for drug delivery applications.
In this article, we describe a simple and effective protocol for synthesizing drug-loaded, disulfide cross-linked micelles based on the self-assembly of a well-defined amphiphilic linear-dendritic copolymer (telodendrimer, TD). TD is composed of polyethylene glycol (PEG) as the hydrophilic segment and a thiolated cholic acid cluster as the core-forming hydrophobic moiety attached stepwise to an amine-terminated PEG using solution-based peptide chemistry. Chemotherapy drugs, such as paclitaxel (PTX), can be loaded using a standard solvent evaporation method. The O2-mediated oxidation was previously utilized to form intra-micellar disulfide cross-links from free thiol groups on the TDs. However, the reaction was slow and not feasible for large-scale production. Recently, an H2O2-mediated oxidation method was explored as a more feasible and efficient approach, and it was 96 times faster than the previously reported method. Using this approach, 50 g of PTX-loaded, disulfide cross-linked nanoparticles have been successfully produced with narrow particle size distribution and high drug loading efficiency. The stability of the resulting micelle solution is analyzed using disrupting conditions such as co-incubation with a detergent, sodium dodecyl sulfate, with or without a reducing agent. The drug-loaded, disulfide cross-linked micelles demonstrated less hemolytic activity when compared to their non-cross-linked counterparts.

To deliver cancer drugs to tumor sites with high specificity and reduced side effects, new methods based on nanoparticles are required. Here, we describe disulfide cross-linked micelles that can be easily prepared by hydrogen peroxide-mediated oxidation and are able to dissociate efficiently under a reducing tumor environment to release payloads.

Nanomedicine is an emerging form of therapy that harnesses the unique properties of particles that are nanometers in scale for biomedical application. ...

A G-quadruplex DNA-affinity Approach for Purification of Enzymatically Active G4 Resolvase1

Higher-order nucleic acid structures called G-quadruplexes (G4s, G4 structures) can form in guanine-rich regions of both DNA and RNA and are highly thermally stable. There are &#62;375,000 putative G4-forming sequences in the human genome, and they are enriched in promoter regions, untranslated regions (UTRs), and within the telomeric repeat. Due to the potential for these structures to affect cellular processes, such as replication and transcription, the cell has evolved enzymes to manage them. One such enzyme is G4 Resolvase 1 (G4R1), which was biochemically co-characterized by our laboratory and Nagamine et al. and found to bind extremely tightly to both G4-DNA and G4-RNA (Kd in the low-pM range). G4R1 is the source of the majority of G4-resolving activity in HeLa cell lysates and has since been implicated to play a role in telomere metabolism, lymph development, gene transcription, hematopoiesis, and immune surveillance. The ability to efficiently express and purify catalytically active G4R1 is of importance for laboratories interested in gaining further insight into the kinetic interaction of G4 structures and G4-resolving enzymes. Here, we describe a detailed method for the purification of recombinant G4R1 (rG4R1). The described procedure incorporates the traditional affinity-based purification of a C-terminal histidine-tagged enzyme expressed in human codon-optimized bacteria with the utilization of the ability of rG4R1 to bind and unwind G4-DNA to purify highly active enzyme in an ATP-dependent elution step. The protocol also includes a quality-control step where the enzymatic activity of rG4R1 is measured by examining the ability of the purified enzyme to unwind G4-DNA. A method is also described that allows for the quantification of purified rG4R1. Alternative adaptations of this protocol are discussed.

G4 Resolvase1 binds to G-quadruplex (G4) structures with the tightest reported affinity for a G4-binding protein and represents the majority of the G4-DNA unwinding activity in HeLa cells. We describe a novel protocol that harnesses the affinity and ATP-dependent unwinding activity of G4-Resolvase1 to specifically purify catalytically active recombinant G4R1.

Higher-order nucleic acid structures called G-quadruplexes (G4s, G4 structures) can form in guanine-rich regions of both DNA and RNA and are highly ...

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 ...

2 in 1: One-step Affinity Purification for the Parallel Analysis of Protein-Protein and Protein-Metabolite Complexes

Cellular processes are regulated by interactions between biological molecules such as proteins, metabolites, and nucleic acids. While the investigation of protein-protein interactions (PPI) is no novelty, experimental approaches aiming to characterize endogenous protein-metabolite interactions (PMI) constitute a rather recent development. Herein, we present a protocol that allows simultaneous characterization of the PPI and PMI of a protein of choice, referred to as bait. Our protocol was optimized for Arabidopsis cell cultures and combines affinity purification (AP) with mass spectrometry (MS)-based protein and metabolite detection. In short, transgenic Arabidopsis lines, expressing bait protein fused to an affinity tag, are first lysed to obtain a native cellular extract. Anti-tag antibodies are used to pull down protein and metabolite partners of the bait protein. The affinity-purified complexes are extracted using a one-step methyl tert-butyl ether (MTBE)/methanol/water method. Whilst metabolites separate into either the polar or the hydrophobic phase, proteins can be found in the pellet. Both metabolites and proteins are then analyzed by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS or UPLC-MS/MS). Empty-vector (EV) control lines are used to exclude false positives. The major advantage of our protocol is that it enables identification of protein and metabolite partners of a target protein in parallel in near-physiological conditions (cellular lysate). The presented method is straightforward, fast, and can be easily adapted to biological systems other than plant cell cultures.

Protein-protein and protein-metabolite interactions are crucial for all cellular functions. Herein, we describe a protocol that allows parallel analysis of these interactions with a protein of choice. Our protocol was optimized for plant cell cultures and combines affinity purification with mass spectrometry-based protein and metabolite detection.

Cellular processes are regulated by interactions between biological molecules such as proteins, metabolites, and nucleic acids. While the investigation of ...

Purification and Reconstitution of TRPV1 for Spectroscopic Analysis

Polymodal ion channels transduce multiple stimuli of different natures into allosteric changes; these dynamic conformations are challenging to determine and remain largely unknown. With recent advances in single-particle cryo-electron microscopy (cryo-EM) shedding light on the structural features of agonist binding sites and the activation mechanism of several ion channels, the stage is set for an in-depth dynamic analysis of their gating mechanisms using spectroscopic approaches. Spectroscopic techniques such as electron paramagnetic resonance (EPR) and double electron-electron resonance (DEER) have been mainly restricted to the study of prokaryotic ion channels that can be purified in large quantities. The requirement for large amounts of functional and stable membrane proteins has hampered the study of mammalian ion channels using these approaches. EPR and DEER offer many advantages, including determination of the structure and dynamic changes of mobile protein regions, albeit at low resolution, that might be difficult to obtain by X-ray crystallography or cryo-EM, and monitoring reversible gating transition (i.e., closed, open, sensitized, and desensitized). Here, we provide protocols for obtaining milligrams of functional detergent-solubilized transient receptor potential cation channel subfamily V member 1 (TRPV1) that can be labeled for EPR and DEER spectroscopy.

This article describes specific methods to obtain biochemical quantities of detergent-solubilized TRPV1 for spectroscopic analysis. The combined protocols provide biochemical and biophysical tools that can be adapted to facilitate structural and functional studies for mammalian ion channels in a membrane-controlled environment.

Polymodal ion channels transduce multiple stimuli of different natures into allosteric changes; these dynamic conformations are challenging to determine ...

Using the Open-Source MALDI TOF-MS IDBac Pipeline for Analysis of Microbial Protein and Specialized Metabolite Data

In order to visualize the relationship between bacterial phylogeny and specialized metabolite production of bacterial colonies growing on nutrient agar, we developed IDBac&#8212;a low-cost and high-throughput matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) bioinformatics pipeline. IDBac software is designed for non-experts, is freely available, and capable of analyzing a few to thousands of bacterial colonies. Here, we present procedures for the preparation of bacterial colonies for MALDI-TOF MS analysis, MS instrument operation, and data processing and visualization in IDBac. In particular, we instruct users how to cluster bacteria into dendrograms based on protein MS fingerprints and interactively create Metabolite Association Networks (MANs) from specialized metabolite data.

IDBac is an open-source mass spectrometry-based bioinformatics pipeline that integrates data from both intact protein and specialized metabolite spectra, collected on cell material scraped from bacterial colonies. The pipeline allows researchers to rapidly organize hundreds to thousands of bacterial colonies into putative taxonomic groups, and further differentiate them based on specialized metabolite production.

In order to visualize the relationship between bacterial phylogeny and specialized metabolite production of bacterial colonies growing on nutrient agar, ...

A Customizable Approach for the Enzymatic Production and Purification of Diterpenoid Natural Products

Diterpenoids form a diverse class of small molecule natural products that are widely distributed across the kingdoms of life and have critical biological functions in developmental processes, interorganismal interactions, and environmental adaptation. Due to these various bioactivities, many diterpenoids are also of economic importance as pharmaceuticals, food additives, biofuels, and other bioproducts. Advanced genomics and biochemical approaches have enabled a rapid increase in the knowledge of diterpenoid-metabolic genes, enzymes, and pathways. However, the structural complexity of diterpenoids and the narrow taxonomic distribution of individual compounds in often only a single species remain constraining factors for their efficient production. Availability of a broader range of metabolic enzymes now provide resources for producing diterpenoids in sufficient titers and purity to facilitate a deeper investigation of this important metabolite group. Drawing on established tools for microbial and plant-based enzyme co-expression, we present an easily operated and customizable protocol for the enzymatic production of diterpenoids in either Escherichia coli or Nicotiana benthamiana, and the purification of desired products via silica chromatography and semi-preparative HPLC. Using the group of maize (Zea mays) dolabralexin diterpenoids as an example, we highlight how modular combinations of diterpene synthase (diTPS) and cytochrome P450 monooxygenase (P450) enzymes can be used to generate different diterpenoid scaffolds. Purified compounds can be used in various downstream applications, such as metabolite structural analyses, enzyme structure-function studies, and in vitro and in planta bioactivity experiments.

Here we present easy to use protocols for producing and purifying diterpenoid metabolites through the combinatorial expression of biosynthetic enzymes in Escherichia coli or Nicotiana benthamiana, followed by chromatographic product purification. The resulting metabolites are suitable for various studies including molecular structure characterization, enzyme functional studies, and bioactivity assays.

Diterpenoids form a diverse class of small molecule natural products that are widely distributed across the kingdoms of life and have critical biological ...

Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

Lipids are structurally diverse amphipathic molecules that are insoluble in water. Lipids are essential contributors to the organization and function of biological membranes, energy storage and production, cellular signaling, vesicular transport of proteins, organelle biogenesis, and regulated cell death. Because the budding yeast Saccharomyces cerevisiae is a unicellular eukaryote amenable to thorough molecular analyses, its use as a model organism helped uncover mechanisms linking lipid metabolism and intracellular transport to complex biological processes within eukaryotic cells. The availability of a versatile analytical method for the robust, sensitive, and accurate quantitative assessment of major classes of lipids within a yeast cell is crucial for getting deep insights into these mechanisms. Here we present a protocol to use liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for the quantitative analysis of major cellular lipids of S. cerevisiae. The LC-MS/MS method described is versatile and robust. It enables the identification and quantification of numerous species (including different isobaric or isomeric forms) within each of the 10 lipid classes. This method is sensitive and allows identification and quantitation of some lipid species at concentrations as low as 0.2 pmol/&#181;L. The method has been successfully applied to assessing lipidomes of whole yeast cells and their purified organelles. The use of alternative mobile phase additives for electrospray ionization mass spectrometry in this method can increase the efficiency of ionization for some lipid species and can be therefore used to improve their identification and quantitation.

Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

We present a protocol using liquid chromatography coupled with tandem mass spectrometry to identify and quantify major cellular lipids in Saccharomyces cerevisiae. The described method for a quantitative assessment of major lipid classes within a yeast cell is versatile, robust, and sensitive.

Lipids are structurally diverse amphipathic molecules that are insoluble in water. Lipids are essential contributors to the organization and function of ...

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 ...

Single Cell Analysis Of Transcriptionally Active Alleles By Single Molecule FISH

Gene transcription is an essential process in cell biology, and allows cells to interpret and respond to internal and external cues. Traditional bulk population methods (Northern blot, PCR, and RNAseq) that measure mRNA levels lack the ability to provide information on cell-to-cell variation in responses. Precise single cell and allelic visualization and quantification is possible via single molecule RNA fluorescence in situ hybridization (smFISH). RNA-FISH is performed by hybridizing target RNAs with labeled oligonucleotide probes. These can be imaged in medium/high throughput modalities, and, through image analysis pipelines, provide quantitative data on both mature and nascent RNAs, all at the single cell level. The fixation, permeabilization, hybridization and imaging steps have been optimized in the lab over many years using the model system described herein, which results in successful and robust single cell analysis of smFISH labeling. The main goal with sample preparation and processing is to produce high quality images characterized by a high signal-to-noise ratio to reduce false positives and provide data that are more accurate. Here, we present a protocol describing the pipeline from sample preparation to data analysis in conjunction with suggestions and optimization steps to tailor to specific samples.&#160;

Single molecule RNA fluorescence in situ hybridization (smFISH) is a method to accurately quantify levels and localization of specific RNAs at the single cell level. Here, we report our validated lab protocols for wet-bench processing, imaging and image analysis for single cell quantification of specific RNAs.

Gene transcription is an essential process in cell biology, and allows cells to interpret and respond to internal and external cues. Traditional bulk ...