This work was supported by NIH Director&#39;s New Innovator Award DP2OD006466 (to K.C.H.). The authors thank Russell Monds for a practical demonstration of the method and for scientific discussions.

1. Grow Bacterial Cultures in 2.5 ml of Media Overnight
Back-dilute cultures 1:100 into 250 ml of fresh media and grow to OD600 of 0.7-0.8. Prepare a solution of 6% sodium dodecyl sulfate (SDS) in water.
CAUTION: SDS powder is hazardous -&#160;avoid inhaling SDS powder; wear a mask over nose and mouth.
2. Day 1 -&#160;Lysing Bacterial Cultures is Performed over the Course of One Day and Overnight
<ol>
	<li>While diluted cultures are growing, set up a boiling water bath on a hot plate in a 1&#160;L beaker. Once the water is boiling, aliquot 6 ml of 6% SDS into 50&#160;ml&#160;polypropylene tubes, add one small stir bar to each tube, secure tube lids to finger-tight, place tubes in water bath, and turn on stirring to 500 rpm on the hot plate.</li>
	<li>Harvest the 250&#160;ml cultures at 5,000 &#215; g for 10 min at room temperature and resuspend pellets in 3 ml&#160;of media or 1x&#160;phosphate-buffered saline. Slowly pipette cell suspensions into 50&#160;ml&#160;tubes with 6% boiling SDS to lyse the cells (final concentration 4% SDS) while the tubes are submerged in the boiling water bath, and reclose the lids to finger-tight. Cell suspensions must be quickly transferred into boiling SDS once resuspended.&#160;Abrupt environmental changes should be avoided, as this could cause erroneous alteration of cell wall structure.</li>
	<li>Cover boiling water bath and allow cells to boil for 3 hr, checking the water level periodically and refilling the water bath when necessary. After 3 hr, turn off the heating element of the hot plate, and continue to stir overnight at 500 rpm.</li>
</ol>
3. Day 2 -&#160;Enzymatic Digestions are Performed over the Course of One Day
<ol>
	<li>If SDS has precipitated in the 50&#160;ml&#160;tubes overnight, set the water bath to boil for an additional 1-2 hr. Turn on a heat block to 60 &#176;C. Prepare a 1 mg/ml stock of Pronase E in 10 mM Tris-HCl (pH 7.2) + 0.06% w/v NaCl and activate Pronase E at 60 &#176;C for at least 30 min.</li>
	<li>Use an ultracentrifuge set at 400,000 &#215; g to spin samples for 20 min at room temperature in order to pellet the large PG polymers and thereby purify them from other cellular components. Remove supernatant carefully, and then resuspend each pellet in room temperature ultrapure water. NOTE: Resuspension volume depends upon the volume of the ultracentrifuge tubes used; use a volume that fills the tubes at least half way but does not exceed the maximum volume of the tubes. Repeat centrifugation/washing until the water does not form bubbles during resuspension, indicating that the SDS has been fully removed (typically three washes). Stop washing the pellet if a white precipitate forms, as this indicates that the sacculi are clumping together. Clumping is not catastrophic, but clumps bind very strongly to plastic and glassware causing large sample loss. In this case, proceed with the protocol using the clumped sacculi sample.</li>
	<li>On the last centrifugation/washing step, resuspend the samples in 900 &#181;l of 10 mM Tris-HCl (pH 7.2) + 0.06% w/v NaCl and transfer to 2&#160;ml tubes previously poked with holes in the tops with a small needle. Add 100 &#181;l of 1 mg/ml activated Pronase E (100 &#181;g/ml final concentration) to each sample and incubate at 60 &#176;C for 2 hr. Set a different heat block to 100 &#176;C.</li>
	<li>Stop the Pronase E digestion by adding 200 &#181;l of 6% SDS to each sample and boil the samples in the 100 &#176;C heat block for 30 min. Set a different heat block to 37 &#176;C and make a 1 mg/ml stock of muramidase (mutanolysin) in 50 mM phosphate buffer (pH 4.9).</li>
	<li>As in step 3.2, use an ultracentrifuge set at 400,000 &#215; g to spin samples for 20 min at room temperature and wash with room temperature ultrapure water until SDS is fully removed (typically three washes). Resuspension volume depends upon the volume of the ultracentrifuge tubes used; use a volume that fills the tubes at least half way but does not exceed the maximum volume of the tubes.</li>
	<li>On the last centrifugation/washing step, resuspend samples in 200 &#181;l of 50 mM sodium phosphate buffer (pH 4.9). This volume can be adjusted according to the amount of peptidoglycan in the sample, and may be species dependent. If there are previously published reports of HPLC analysis for the species of interest, this volume can be estimated based on these quantitations (a compilation of HPLC PG studies can be found in the Supplemental Information of reference7). For other species, the sacculus prep can be executed up to this step and then different amounts of resuspension volumes can be added to replicate samples in order to determine the minimal volume that allows the PG to remain in solution (see Discussion for further estimates). If the sample contains more peptidoglycan, increase the resuspension volume; if the sample has little peptidoglycan, reduce the resuspension volume to a minimum of 50 &#181;l.</li>
	<li>Transfer samples to 1.5&#160;ml tubes and add 1 mg/ml muramidase to give a final concentration of 40 &#181;g/ml. Incubate 6-8 hr or overnight at 37 &#176;C.</li>
</ol>
4. Day 3 -&#160;Preparation of Samples for UPLC is Performed on the Last Day
<ol>
	<li>Turn on a heat block to 100 &#176;C. Boil the samples without SDS for 5 min to stop the muramidase digestion. Centrifuge samples for 10 min at 16,000 &#215; g at room temperature, then transfer the supernatant (muropeptides are now soluble) to 13 mm x&#160;100&#160;mm glass tubes. Try to recover as much supernatant as possible, getting very close to the pellet without disturbing it.</li>
	<li>Adjust the pH by adding 500 mM borate buffer (pH 9) to the sample for a final concentration of 100 mM borate buffer. Borate buffer is compatible with the reducing agent sodium borohydride. Add 1-2 grains of sodium borohydride to reduce each sample and let the reaction proceed for at least 30 min at room temperature. CAUTION: Sodium borohydride is highly reactive and dangerous to handle -&#160;avoid contact with skin (wear gloves) and avoid contact with eyes (wear safety glasses).</li>
	<li>Adjust samples to pH 3-4 (the muropeptide isoelectric point is ~3.5) with 50% v/v orthophosphoric acid using 20&#160;&#181;l increments until pH 6, as measured with pH indicator paper, then using 2&#160;&#181;l&#160;increments. CAUTION: Orthophosphoric acid is corrosive and dangerous to handle -&#160;avoid contact with skin (wear gloves) and avoid contact with eyes (wear safety glasses). The sample should bubble in response to addition of orthophosphoric acid; when the sample stops bubbling, this typically indicates a pH of 6 has been reached. If no bubbling occurs in the sample, this may indicate that the amount of sodium borohydride added was too small. In this case, stop lowering pH, carefully add one or two grains of sodium borohydride, let react for 5-10 min, then resume pH adjustment.</li>
	<li>Filter sample through a 0.22&#160;&#181;m syringe filter directly into a UPLC vial. If a precipitate has formed, heat the tube with several passes through a flame before filtering. If the sample will not be injected into the UPLC instrument within the day, freeze at -20 &#176;C overnight. Samples can be stored for up to a year at -80 &#176;C. The sample can be thawed by passing through a flame several times.</li>
	<li>Inject 10 &#181;l&#160;of each sample onto a UPLC instrument equipped with a C18 1.7&#160;&#181;m reversed-phase column and an absorbance detector set to monitor 202-208 nm. Samples are injected sequentially, but autosampler capabilities allow up to 96 samples to be processed in batch. Use 50 mM sodium phosphate (pH 4.35) +&#160;0.4% v/v sodium azide for solvent A, and 75 mM sodium phosphate (pH 4.95) + 15% v/v methanol for solvent B. NOTE: Sodium azide is added to compensate for the 205 nm absorption of methanol to avoid baseline drift. Set the flow to 0.25 ml/min and use a linear gradient over 25 min to achieve 100% solvent B and sequential elution of muropeptides within 30 min. If mass spectrometry will be used to characterize muropeptides after UPLC, collect fractions of the peak of interest in a fraction collector and dry the fractions using a centrifugal evaporator.</li>
</ol>

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A., Vollmer, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+the+regulation+of+peptidoglycan+synthesis+to+bacterial+growth+and+morphology.">From the regulation of peptidoglycan synthesis to bacterial growth and morphology.</a> Nat. Rev. Microbiol. 10, 123-136 (2011).</li><li>Vollmer, W., Blanot, D., de Pedro, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peptidoglycan+structure+and+architecture.">Peptidoglycan structure and architecture.</a> FEMS Microbiol. Rev. 32, 149-167 (2008).</li><li>Davis, K. M., Weiser, J. 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V., Labischinski, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The target of penicillin: the murein sacculus of bacterial cell walls architecture and growth. , (1983).</li><li>Boylen, C. W., Ensign, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ratio+of+teichoic+acid+and+peptidoglycan+in+cell+walls+of+Bacillus+subtilis+following+spire+germination+and+during+vegetative+growth.">Ratio of teichoic acid and peptidoglycan in cell walls of Bacillus subtilis following spire germination and during vegetative growth.</a> J. Bacteriol. 96, 421-427 (1968).</li><li>Sutow, A. B., Welker, N. 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Production costs for this article&#160;were sponsored by Waters Corporation.

A critical step in this procedure is step 3.1 of the second day of sample preparation. If the SDS has precipitated overnight, or if the samples have been stored in 4% SDS for several weeks at room temperature, the samples must be reboiled for at least 1 hr to redissolve the SDS. A common cause for SDS precipitation is the use of media with potassium salts, so potassium should be avoided in media if possible. As mentioned in the Representative Results section, it is also critical to adjust the pH to within the isoelectric...

The goal of the method described herein is to isolate intact bacterial cell walls (sacculi) and to digest the peptidoglycan (PG) such that Ultra Performance Liquid Chromatography (UPLC) can be used to provide information such as the identity of the muropeptide components and their concentrations, the average length of glycan strands, and the fraction of material involved in crosslinks between strands. For a detailed discussion of PG biochemistry and muropeptide species, there are several excellent reviews that describe PG structure and its role in infection, resistance, morphogenesis, and growth1-6. High Performance Liquid Chromatography (HPLC) for PG analysis was initially developed by Glauner and Schwarz in the 1980s, and more recently has been enhanced and applied extensively in the labs of Miguel de Pedro and Waldemar Vollmer. Previous methods utilized amino acid analysis or paper chromatography, time-consuming and tedious techniques that do not yield accurate or complete assessments of cell-wall components.
UPLC analysis can be easily implemented in any basic research laboratory that has access to an ultracentrifuge and UPLC. The UPLC method that we present below isolates complete sacculi, thereby providing comprehensive, quantitative information on all chemical species therein. This method yields precise quantification of all muropeptides across a population of bacteria, all within a 20 min UPLC run. The implementation of this method involves only basic laboratory skills, without significant financial investment in materials. In order to execute the steps in this method, researchers need only be skilled in pipetting, preparing buffers and enzymes, and adjusting pH, making it accessible to a wide range of scientific disciplines. The choice of enzymes used in this protocol depends on the species of bacterium being analyzed; the protocol described here is useful for Escherichia coli, and has generally been found to be adequate for isolating sacculi from other Gram-negative organisms. Consultation with the literature is recommended when applying this method to Gram-positive bacteria; in these species, sacculus purification has traditionally been more difficult. In particular, this method may have to be altered in terms of enzyme choice and length of digestion times to accommodate the thicker walls and accessory polymers such as teichoic acids of Gram-positive bacteria. The first enzyme in this protocol cleaves outer membrane lipoprotein (such as Braun&#8217;s lipoprotein, or Lpp) attachment to the peptidoglycan, thereby releasing all but the C-terminal di- (or tri-) peptide of the Lpp from the cell wall. This step is necessary when examining Enterobacteria, but many other Gram-negative bacteria have no Lpp equivalents, and therefore this step can be skipped. A second enzyme specifically cleaves after the muramic acid component of the peptidoglycan, solubilizing the disaccharide subunit that forms the muropeptide species. To provide an accurate assessment of the architecture of the PG, care should be taken in digesting the sacculi to prevent cleavage of the crossbridges or any other part of the peptide stem.
Although the chemical compositions of peptidoglycan from over 100 strains of ~40 bacterial species have been analyzed by HPLC, no analyses have been performed with UPLC technology. In addition, previous work has characterized peptidoglycan from only a small fraction of the bacterial domain, in part limited by the throughput of HPLC. Therefore, dissemination of this method to as many researchers as possible, and implementation on UPLC platforms, will be critical for driving physiological studies of the large fraction of bacterial species whose peptidoglycan has yet to be categorized.

<table border="0" cellpadding="0" cellspacing="0" width="640">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:240px;">Pronase E</td>
			<td style="width:115px;">Amresco</td>
			<td style="width:119px;">E629</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mutanolysin from Streptomyces</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:119px;">M9901</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Sodium borohydride (NaBH4)&#160;</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:119px;">452882</td>
			<td>Sodium borohydride is highly reactive and dangerous to handle</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Orthophosphoric acid&#160;</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:119px;">79607</td>
			<td>Orthophosphoric acid is corrosive and dangerous to handle</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Boric acid</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:119px;">31146</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium azide</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:119px;">S2002</td>
			<td>Sodium azide is a poison</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium tetraborate</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:119px;">221732</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Millex 0.22 &#956;m syringe filters</td>
			<td style="width:115px;">Fisher</td>
			<td style="width:119px;">SLGVR04NL</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pH strips (pH range 0-6)</td>
			<td style="width:115px;">Fisher</td>
			<td style="width:119px;">M95863</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">50 ml polypropylene Falcon tubes</td>
			<td style="width:115px;">VWR</td>
			<td style="width:119px;">21008-951</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">13 mm x 100 mm glass tubes</td>
			<td style="width:115px;">Kimble Chase</td>
			<td style="width:119px;">60CM13</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:240px;">12 mm x 32 mm screw neck glass recovery vial</td>
			<td style="width:115px;">Waters</td>
			<td style="width:119px;">186000327C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:240px;">Sodium Dodecyl Sulfate</td>
			<td style="width:115px;">Ambion</td>
			<td style="width:119px;">AM9820</td>
			<td>SDS powder is hazardous</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:240px;"></td>
			<td style="width:115px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:240px;">Instrumentation</td>
			<td style="width:115px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td colspan="2" height="21" style="height:21px;">Waters Acquity UPLC H-Class system, including:</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td colspan="2" height="21" style="height:21px;">Acquity UPLC H-Class Sample Manager FTN</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td colspan="2" height="21" style="height:21px;">Acquity UPLC H-Class Quaternary Solvent Manager</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td colspan="2" height="21" style="height:21px;">Acquity UPLC BEH C18 1.7 &#181;m column</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Acquity UPLC PDA Detector</td>
			<td style="width:115px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Waters Fraction Collector III</td>
			<td style="width:115px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td colspan="2" height="21" style="height:21px;">Acquity UPLC 30 cm Column Heater/Cooler</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
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isolation and preparation of bacterial cell walls for compositional analysis by ultra performance liquid chromatography

The bacterial cell wall is critical for the determination of cell shape during growth and division, and maintains the mechanical integrity of cells in the face of turgor pressures several atmospheres in magnitude. Across the diverse shapes and sizes of the bacterial kingdom, the cell wall is composed of peptidoglycan, a macromolecular network of sugar strands crosslinked by short peptides. Peptidoglycan&#8217;s central importance to bacterial physiology underlies its use as an antibiotic target and has motivated genetic, structural, and cell biological studies of how it is robustly assembled during growth and division. Nonetheless, extensive investigations are still required to fully characterize the key enzymatic activities in peptidoglycan synthesis and the chemical composition of bacterial cell walls. High Performance Liquid Chromatography (HPLC) is a powerful analytical method for quantifying differences in the chemical composition of the walls of bacteria grown under a variety of environmental and genetic conditions, but its throughput is often limited. Here, we present a straightforward procedure for the isolation and preparation of bacterial cell walls for biological analyses of peptidoglycan via HPLC and Ultra Performance Liquid Chromatography (UPLC), an extension of HPLC that utilizes pumps to deliver ultra-high pressures of up to 15,000 psi, compared with 6,000 psi for HPLC. In combination with the preparation of bacterial cell walls presented here, the low-volume sample injectors, detectors with high sampling rates, smaller sample volumes, and shorter run times of UPLC will enable high resolution and throughput for novel discoveries of peptidoglycan composition and fundamental bacterial cell biology in most biological laboratories with access to an ultracentrifuge and UPLC.

Using the procedure outlined in Figure&#160;1, the final sample should consist of at least 200 &#181;l&#160;of clear solution that has been filtered directly into a UPLC vial (step 4.4). UPLC separation of the various muropeptides in a bacterial sample relies upon their relative solubility between the liquid mobile phase and the column&#8217;s stationary phase. Reversed-phase C18 columns provide a strongly hydrophobic matrix to separate the muropeptide species based on hydrophobicity and size8...

Watch this Scientific Journal Video about Isolation and Preparation of Bacterial Cell Walls for Compositional Analysis by Ultra Performance Liquid Chromatography at JoVE.com

Isolation and Preparation of Bacterial Cell Walls for Compositional Analysis by Ultra Performance Liquid Chromatography

The bacterial cell wall is composed of peptidoglycan, a macromolecular network of sugar strands crosslinked by peptides. Ultra Performance Liquid Chromatography provides high resolution and throughput for novel discoveries of peptidoglycan composition. We present a procedure for the isolation of cell walls (sacculi) and their subsequent preparation for analysis via UPLC.

The bacterial cell wall is critical for the determination of cell shape during growth and division, and maintains the mechanical integrity of cells in the ...

isolation-preparation-bacterial-cell-walls-for-compositional-analysis

Research

JoVE Journal

Chemistry

16.1K vues.  Stanford University. La paroi cellulaire bactérienne est composée de peptidoglycane, un réseau macromoléculaire de brins de sucre réticulés par des peptides. La chromatographie liquide ultra-performante offre une résolution et un débit élevés pour de nouvelles découvertes de composition de peptidoglycane. Nous présentons un procédé pour l’isolement des murs de cellules (sacculi) et leur préparation suivante pour l’analyse par l’intermédiaire d’UPLC.

Vidéo: Isolation and Preparation of Bacterial Cell Walls for Compositional Analysis by Ultra Performance Liquid Chromatography

Large Scale Non-targeted Metabolomic Profiling of Serum by Ultra Performance Liquid Chromatography-Mass Spectrometry (UPLC-MS)

Non-targeted metabolite profiling by ultra performance liquid chromatography coupled with mass spectrometry (UPLC-MS) is a powerful technique to investigate metabolism. The approach offers an unbiased and in-depth analysis that can enable the development of diagnostic tests, novel therapies, and further our understanding of disease processes. The inherent chemical diversity of the metabolome creates significant analytical challenges and there is no single experimental approach that can detect all metabolites. Additionally, the biological variation in individual metabolism and the dependence of metabolism on environmental factors necessitates large sample numbers to achieve the appropriate statistical power required for meaningful biological interpretation. To address these challenges, this tutorial outlines an analytical workflow for large scale non-targeted metabolite profiling of serum by UPLC-MS. The procedure includes guidelines for sample organization and preparation, data acquisition, quality control, and metabolite identification and will enable reliable acquisition of data for large experiments and provide a starting point for laboratories new to non-targeted metabolite profiling by UPLC-MS.

Large Scale Non-targeted Metabolomic Profiling of Serum by Ultra Performance Liquid Chromatography-Mass Spectrometry UPLC-MS

Non-targeted metabolite profiling by ultra performance liquid chromatography coupled with mass spectrometry (UPLC-MS) is a powerful technique to investigate metabolism. This article outlines a typical workflow utilized for non-targeted metabolite profiling of serum including sample organization and preparation, data acquisition, data analysis, quality control, and metabolite identification.

Non-targeted metabolite profiling by ultra performance liquid chromatography coupled with mass spectrometry (UPLC-MS) is a powerful technique to ...

Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry (UPLC-HRMS)

Here we present a workflow to analyze the metabolic profiles for biological samples of interest including; cells, serum, or tissue. The sample is first separated into polar and non-polar fractions by a liquid-liquid phase extraction, and partially purified to facilitate downstream analysis. Both aqueous (polar metabolites) and organic (non-polar metabolites) phases of the initial extraction are processed to survey a broad range of metabolites. Metabolites are separated by different liquid chromatography methods based upon their partition properties. In this method, we present microflow ultra-performance (UP)LC methods, but the protocol is scalable to higher flows and lower pressures. Introduction into the mass spectrometer can be through either general or compound optimized source conditions. Detection of a broad range of ions is carried out in full scan mode in both positive and negative mode over a broad m/z range using high resolution on a recently calibrated instrument. Label-free differential analysis is carried out on bioinformatics platforms. Applications of this approach include metabolic pathway screening, biomarker discovery, and drug development.

Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry UPLC-HRMS

Untargeted metabolomics provides a hypothesis generating snapshot of a metabolic profile. This protocol will demonstrate the extraction and analysis of metabolites from cells, serum, or tissue. A range of metabolites are surveyed using liquid-liquid phase extraction, microflow ultraperformance liquid chromatography/high-resolution mass spectrometry (UPLC-HRMS) coupled to differential analysis software.

Here we present a workflow to analyze the metabolic profiles for biological samples of interest including; cells, serum, or tissue. The sample is first ...

The Use of Gas Chromatography to Analyze Compositional Changes of Fatty Acids in Rat Liver Tissue during Pregnancy

Gas chromatography (GC) is a highly sensitive method used to identify and quantify the fatty acid content of lipids from tissues, cells, and plasma/serum, yielding results with high accuracy and high reproducibility. In metabolic and nutrition studies GC allows assessment of changes in fatty acid concentrations following interventions or during changes in physiological state such as pregnancy. Solid phase extraction (SPE) using aminopropyl silica cartridges allows separation of the major lipid classes including triacylglycerols, different phospholipids, and cholesteryl esters (CE). GC combined with SPE was used to analyze the changes in fatty acid composition of the CE fraction in the livers of virgin and pregnant rats that had been fed various high and low fat diets. There are significant diet/pregnancy interaction effects upon the omega-3 and omega-6 fatty acid content of liver CE, indicating that pregnant females have a different response to dietary manipulation than is seen among virgin females.

Pregnancy leads to significant changes to the fatty acid composition of maternal tissues. Lipid profiles can be obtained via gas chromatography to allow identification and quantification of fatty acids in individual lipid classes among rats fed various high and low fat diets during pregnancy.

Gas chromatography (GC) is a highly sensitive method used to identify and quantify the fatty acid content of lipids from tissues, cells, and plasma/serum, ...

Synthesis and Catalytic Performance of Gold Intercalated in the Walls of Mesoporous Silica

As a promising catalytically active nano reactor, gold nanoparticles intercalated in mesoporous silica (GMS) were successfully synthesized and properties of the materials were investigated. We used a one pot sol-gel approach to intercalate gold nano particles in the walls of mesoporous silica. To start with the synthesis, P123 was used as template to form micelles. Then TESPTS was used as a surface modification agent to intercalate gold nano particles. Following this process, TEOS was added in as a silica source which underwent a polymerization process in acid environment. After hydrothermal processing and calcination, the final product was acquired. Several techniques were utilized to characterize the porosity, morphology and structure of the gold intercalated mesoporous silica. The results showed a stable structure of mesoporous silica after gold intercalation. Through the oxidation of benzyl alcohol as a benchmark reaction, the GMS materials showed high selectivity and recyclability.

Here, we present a protocol with a sol-gel process to synthesize gold intercalated in the walls of mesoporous materials (GMS), which is confirmed to possess a mesoporous matrix with gold intercalated in the walls imparting great stability and recyclability.

As a promising catalytically active nano reactor, gold nanoparticles intercalated in mesoporous silica (GMS) were successfully synthesized and properties ...

Post Column Derivatization Using Reaction Flow High Performance Liquid Chromatography Columns

A protocol for the use of reaction flow high performance liquid chromatography columns for methods employing post column derivatization (PCD) is presented. A major difficulty in adapting PCD to modern HPLC systems and columns is the need for large volume reaction coils that enable reagent mixing and then the derivatization reaction to take place. This large post column dead volume leads to band broadening, which results in a loss of observed separation efficiency and indeed detection in sensitivity. In reaction flow post column derivatization (RF-PCD) the derivatization reagent(s) are pumped against the flow of mobile phase into either one or two of the outer ports of the reaction flow column where it is mixed with column effluent inside a frit housed within the column end fitting. This technique allows for more efficient mixing of the column effluent and derivatization reagent(s) meaning that the volume of the reaction loops can be minimized or even eliminated altogether. It has been found that RF-PCD methods perform better than conventional PCD methods in terms of observed separation efficiency and signal to noise ratio. A further advantage of RF-PCD techniques is the ability to monitor effluent coming from the central port in its underivatized state. RF-PCD has currently been trialed on a relatively small range of post column reactions, however, there is currently no reason to suggest that RF-PCD could not be adapted to any existing one or two component (as long as both reagents are added at the same time) post column derivatization reaction.

A protocol for the use of reaction flow high performance liquid chromatography columns for methods employing post column derivatization (PCD) is presented.

A protocol for the use of reaction flow high performance liquid chromatography columns for methods employing post column derivatization (PCD) is ...

Radiolabeling and Quantification of Cellular Levels of Phosphoinositides by High Performance Liquid Chromatography-coupled Flow Scintillation

Phosphoinositides (PtdInsPs) are essential signaling lipids responsible for recruiting specific effectors and conferring organelles with molecular identity and function. Each of the seven PtdInsPs varies in their distribution and abundance, which are tightly regulated by specific kinases and phosphatases. The abundance of PtdInsPs can change abruptly in response to various signaling events or disturbance of the regulatory machinery. To understand how these events lead to changes in the amount of PtdInsPs and their resulting impact, it is important to quantify PtdInsP levels before and after a signaling event or between control and abnormal conditions. However, due to their low abundance and similarity, quantifying the relative amounts of each PtdInsP can be challenging. This article describes a method for quantifying PtdInsP levels by metabolically labeling cells with 3H-myo-inositol, which is incorporated into PtdInsPs. Phospholipids are then precipitated and deacylated. The resulting soluble 3H-glycero-inositides are further extracted, separated by high-performance liquid chromatography (HPLC), and detected by flow scintillation. The labeling and processing of yeast samples is described in detail, as well as the instrumental setup for the HPLC and flow scintillator. Despite losing structural information regarding acyl chain content, this method is sensitive and can be optimized to concurrently quantify all seven PtdInsPs in cells.

Phosphoinositides are signaling lipids whose relative abundance rapidly changes in response to various stimuli. This article describes a method to measure the abundance of phosphoinositides by metabolically labeling cells with 3H-myo-inositol, followed by extraction and deacylation. Extracted glycero-inositides are then separated by high-performance liquid chromatography and quantified by flow scintillation.

Phosphoinositides (PtdInsPs) are essential signaling lipids responsible for recruiting specific effectors and conferring organelles with molecular ...

Sequencing of Plant Wall Heteroxylans Using Enzymic, Chemical (Methylation) and Physical (Mass Spectrometry, Nuclear Magnetic Resonance) Techniques

This protocol describes the specific techniques used for the characterization of reducing end (RE) and internal region glycosyl sequence(s) of heteroxylans. De-starched wheat endosperm cell walls were isolated as an alcohol-insoluble residue (AIR)1 and sequentially extracted with water (W-sol Fr) and 1 M KOH containing 1% NaBH4 (KOH-sol Fr) as described by Ratnayake et al. (2014)2. Two different approaches (see summary in Figure 1) are adopted. In the first, intact W-sol AXs are treated with 2AB to tag the original RE backbone chain sugar residue and then treated with an endoxylanase to generate a mixture of 2AB-labelled RE and internal region reducing oligosaccharides, respectively. In a second approach, the KOH-sol Fr is hydrolyzed with endoxylanase to first generate a mixture of oligosaccharides which are subsequently labelled with 2AB. The enzymically released ((un)tagged) oligosaccharides from both W- and KOH-sol Frs are then methylated and the detailed structural analysis of both the native and methylated oligosaccharides is performed using a combination of MALDI-TOF-MS, RP-HPLC-ESI-QTOF-MS and ESI-MSn. Endoxylanase digested KOH-sol AXs are also characterized by nuclear magnetic resonance (NMR) that also provides information on the anomeric configuration. These techniques can be applied to other classes of polysaccharides using the appropriate endo-hydrolases.

Sequencing of Plant Wall Heteroxylans Using Enzymic, Chemical Methylation and Physical Mass Spectrometry, Nuclear Magnetic Resonance Techniques

This protocol describes the specific techniques used for the structural characterization of reducing end (RE) and internal region glycosyl sequence(s) of heteroxylans by tagging the RE with 2 aminobenzamide prior to enzymatic (endoxylanase) hydrolysis and then analysis of the resultant oligosaccharides using mass spectrometry (MS) and nuclear magnetic resonance (NMR).

This protocol describes the specific techniques used for the characterization of reducing end (RE) and internal region glycosyl sequence(s) of ...

An HS-MRM Assay for the Quantification of Host-cell Proteins in Protein Biopharmaceuticals by Liquid Chromatography Ion Mobility QTOF Mass Spectrometry

The analysis of low-level (1-100 ppm) protein impurities (e.g., host-cell proteins (HCPs)) in protein biotherapeutics is a challenging assay requiring high sensitivity and a wide dynamic range. Mass spectrometry-based quantification assays for proteins typically involve protein digestion followed by the selective reaction monitoring/multiple reaction monitoring (SRM/MRM) quantification of peptides using a low-resolution (Rs ~1,000) tandem quadrupole mass spectrometer. One of the limitations of this approach is the interference phenomenon observed when the peptide of interest has the "same" precursor and fragment mass (in terms of m/z values) as other co-eluting peptides present in the sample (within a 1-Da window). To avoid this phenomenon, we propose an alternative mass spectrometric approach, a high selectivity (HS) MRM assay that combines the ion mobility separation of peptide precursors with the high-resolution (Rs ~30,000) MS detection of peptide fragments. We explored the capabilities of this approach to quantify low-abundance peptide standards spiked in a monoclonal antibody (mAb) digest and demonstrated that it has the sensitivity and dynamic range (at least 3 orders of magnitude) typically achieved in HCP analysis. All six peptide standards were detected at concentrations as low as 0.1 nM (1 femtomole loaded on a 2.1-mm ID chromatographic column) in the presence of a high-abundance peptide background (2 &#181;g of a mAb digest loaded on-column). When considering the MW of rabbit phosphorylase (97.2 kDa), from which the spiked peptides were derived, the LOQ of this assay is lower than 50 ppm. Relative standard deviations (RSD) of peak areas (n = 4 replicates) were less than 15% across the entire concentration range investigated (0.1-100 nM or 1-1,000 ppm) in this study.

Here, we describe a chromatographic assay coupled with the ion mobility separation of peptide precursors followed by the high-resolution (~30,000) MS-detection of peptide fragments for the quantification of spiked peptide standards in a monoclonal antibody digest.

The analysis of low-level (1-100 ppm) protein impurities (e.g., host-cell proteins (HCPs)) in protein biotherapeutics is a challenging assay requiring ...

A Straightforward Method for Glucosinolate Extraction and Analysis with High-pressure Liquid Chromatography (HPLC)

Glucosinolates are a well-studied and highly diverse class of natural plant compounds. They play important roles in plant resistance, rapeseed oil quality, food flavoring, and human health. The biological activity of glucosinolates is released upon tissue damage, when they are mixed with the enzyme myrosinase. This results in the formation of pungent and toxic breakdown products, such as isothiocyanates and nitriles. Currently, more than 130 structurally different glucosinolates have been identified. The chemical structure of the glucosinolate is an important determinant of the product that is formed, which in turn determines its biological activity. The latter may range from detrimental (e.g., progoitrin) to beneficial (e.g., glucoraphanin). Each glucosinolate-containing plant species has its own specific glucosinolate profile. For this reason, it is important to correctly identify and reliably quantify the different glucosinolates present in brassicaceous leaf, seed, and root crops or, for ecological studies, in their wild relatives. Here, we present a well-validated, targeted, and robust method to analyze glucosinolate profiles in a wide range of plant species and plant organs. Intact glucosinolates are extracted from ground plant materials with a methanol-water mixture at high temperatures to disable myrosinase activity. Thereafter, the resulting extract is brought onto an ion-exchange column for purification. After sulfatase treatment, the desulfoglucosinolates are eluted with water and the eluate is freeze-dried. The residue is taken up in an exact volume of water, which is analyzed by high-pressure liquid chromatography (HPLC) with a photodiode array (PDA) or ultraviolet (UV) detector. Detection and quantification are achieved by conducting comparisons of the retention times and UV spectra of commercial reference standards. The concentrations are calculated based on a sinigrin reference curve and well-established response factors. The advantages and disadvantages of this straightforward method, when compared to faster and more technologically advanced methods, are discussed here.

A Straightforward Method for Glucosinolate Extraction and Analysis with High-pressure Liquid Chromatography HPLC

Here, we describe in great detail an established and robust protocol for the extraction of glucosinolates from ground plant materials. After an on-column sulfatase treatment of the extracts, the desulfoglucosinolates are eluted and analyzed by high-pressure liquid chromatography.

Glucosinolates are a well-studied and highly diverse class of natural plant compounds. They play important roles in plant resistance, rapeseed oil ...

Liquid-cell Transmission Electron Microscopy for Tracking Self-assembly of Nanoparticles

Drying a nanoparticle dispersion is a versatile way to create self-assembled structures of nanoparticles, but the mechanism of this process is not fully understood. We have traced the trajectories of individual nanoparticles using liquid-cell transmission electron microscopy (TEM) to investigate the mechanism of the assembly process. Herein, we present the protocols used for liquid-cell TEM studies of the self-assembly mechanism. First, we introduce the detailed synthetic protocols used to produce uniformly sized platinum and lead selenide nanoparticles. Next, we present the microfabrication processes used to produce liquid cells with silicon nitride or silicon windows and then describe the loading and imaging procedures of the liquid-cell TEM technique. Several notes are included to provide helpful tips for the entire process, including how to manage the fragile cell windows. The individual motions of nanoparticles tracked by liquid-cell TEM revealed that changes in the solvent boundaries caused by evaporation affected the self-assembly process of nanoparticles. The solvent boundaries drove nanoparticles to primarily form amorphous aggregates, followed by flattening of the aggregates to produce a 2-dimensional (2D) self-assembled structure. These behaviors are also observed for different nanoparticle types and different liquid-cell compositions.

Here we introduce experimental protocols for the real-time observation of a self-assembly process using liquid-cell transmission electron microscopy.

Drying a nanoparticle dispersion is a versatile way to create self-assembled structures of nanoparticles, but the mechanism of this process is not fully ...