We thank all the members of the Shonai Regional Industry Promotion Center for their help. This work was supported in part by research funds from Yamagata Prefecture and Tsuruoka City, by the National Cancer Center Research and Development Fund [grant number 28-A-9], and by the Japan Society for the Promotion of Science (JSPS) KAKENHI [grant number 17K07189] to HM.

1. Cell culture on day 1
NOTE: Each sample for metabolite extraction should be prepared from a single 100 mm tissue culture dish that is moderately but not fully confluent (containing approximately 2&#8211;5 million cells). Calculate the number of dishes needed for the assay and prepare them accordingly.
<ol>
	<li>Culture HCC827 and PC-9 cells in 5% CO2 at 37 &#176;C in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS).</li>
	<li>Aspirate the cell culture media from the 100 mm culture dishes.</li>
	<li>Wash cells on each dish using 2 mL of phosphate buffered saline (PBS) solution without calcium and magnesium. Gently rock each dish so that the PBS solution completely covers the surface of the dish.</li>
	<li>Aspirate the wash buffer from the culture dishes.</li>
	<li>Warm 0.25% trypsin-EDTA solution to 37 &#176;C and add 2 mL of trypsin-EDTA solution with a 5 mL serological pipette. Gently rock each dish so that the trypsin completely covers the surface of the dish.</li>
	<li>Incubate the culture dishes at 37 &#176;C for approximately 5 min.</li>
	<li>Add 4 mL of pre-warmed complete growth medium per dish. Resuspend the cells in medium by gently pipetting several times.</li>
	<li>Transfer each cell suspension to a separate 15 mL conical tube and centrifuge at 800 &#215; g for 5 min.</li>
	<li>Resuspend each cell pellet in 2 mL of pre-warmed complete growth medium.</li>
	<li>Determine the total number and percent viability of the cells using the automated cell counter and 0.4% trypan blue solution.
	<ol>
		<li>Mix 10 &#956;L of cell suspension and 10 &#956;L of 0.4% trypan blue solution.</li>
		<li>Load 10 &#956;L of sample into the cell counting chamber slide through capillary action.</li>
		<li>Insert the chamber slide into the automated cell counter. The transmitted light automatically illuminates and the instrument auto focuses on the cell.</li>
		<li>Press the capture button to capture the image and display the results.</li>
		<li>If necessary, add further growth medium to obtain the desired cell concentration.</li>
	</ol>
	</li>
	<li>Seed approximately 1&#8211;2.5 million cells per 100 mm cell culture dish. 
	NOTE: Metabolite concentrations determined by CE-MS analysis will be normalized based on the number of viable cells. For the purpose of cell counting, it is necessary to prepare at least one extra seeded culture dish for each group.</li>
	<li>Incubate the culture dishes in 5% CO2 at 37 &#176;C for 18 h.</li>
</ol>
2. Preparation of reagents
<ol>
	<li>Dilute a commercial internal standard solution including L-methionine sulfone and d-camphor-10-sulfonic acid 1000-fold in ultrapure water. 
	NOTE: For fewer than 80 samples, simply mix 50 &#956;L of the Internal Standard Solution 1 and 45 mL of ultrapure water in a 50 mL volumetric flask, then bring the solution up to 50 mL with ultrapure water.</li>
	<li>Prepare a 0.05 g/mL mannitol solution in ultrapure water as wash buffer. 
	NOTE: For fewer than 30 samples, simply dissolve 25 g of mannitol in 500 mL of ultrapure water. Approximately 15 mL of wash buffer is required per 100 mm culture dish, so prepare a sufficient volume of wash buffer according to the number of samples.</li>
</ol>
3. Pre-washing centrifugal filter units
<ol>
	<li>Pipette 250 &#956;L of ultrapure water into the filter cup of each centrifugal filter unit (see the Table of Materials). 
	NOTE: Two filter units per sample are required.</li>
	<li>Cap the filter units tightly and centrifuge at 9,100 &#215; g at 4 &#176;C for 5 min.</li>
	<li>Check the volume of each filtrate&#8212;if significant filtrate has accumulated during the first short spin, the filter unit may be defective. In this case, discard the filter unit and use a new filter unit instead.</li>
	<li>Close the lids of the filter units tightly and centrifuge again at 9,100 &#215; g at 4 &#176;C for 30 min.</li>
	<li>Ensure that no ultrapure water remains in any of the filter cups; remove the filtered ultrapure water in each collection tube with a pipette and discard. 
	NOTE: Do not try to remove residual water in a filter cup with a pipette as it may damage the filter.</li>
	<li>Replace the filter cups into their collection tubes. 
	NOTE: Use the centrifugal filter units within an hour since the filters may become damaged upon drying.</li>
</ol>
4. Cell culture on day 2
<ol>
	<li>Take out the 100 mm culture dishes from the incubator. 
	NOTE: The recommended cell culture duration is 18 h.</li>
	<li>Aspirate the cell culture medium from each 100 mm culture dish.</li>
	<li>Add 10 mL of cell culture medium that includes the appropriate concentrations of compounds or drugs to each dish, taking care to not disturb the cell layer. 
	NOTE: For demonstration purposes, we added 10 &#956;L of 250 mM diamide dissolved in PBS (final concentration of 250 &#956;M) in this experiment.</li>
	<li>Incubate the culture dishes at 37 &#176;C for 30 min in the presence of diamide or PBS as a control.</li>
	<li>Aspirate the cell culture medium from each 100 mm culture dish.</li>
	<li>Wash cells by gently adding 2 mL of 5% mannitol solution to the edge of each dish, taking care to not to disturb the cell layer, then slightly tilt the dish. 
	NOTE: PBS or saline solution interferes with CE-MS-based metabolomic analysis and adversely affects measurement results, and thus should not be used as wash buffer.</li>
	<li>Aspirate the wash buffer from each culture dish, then wash cells again by gently adding 10 mL of wash buffer per dish and slightly tilting the dish.</li>
	<li>Completely aspirate the wash buffer from the edge of each culture dish. 
	NOTE: Aspirate as much wash buffer as possible, while paying attention to not aspirate the cells. Residual mannitol may interfere with CE-MS analysis; aspiration of cells will decrease the number of cells and thus become a source of error in data normalization.</li>
</ol>
5. Extraction of metabolites from cultured cells
<ol>
	<li>Add 800 &#956;L of 99.7% methanol per culture dish. Gently rock each culture dish back and forth to cover its entire surface. Leave the dishes at room temperature for 30 s.</li>
	<li>Slowly add 550 &#956;L of the diluted internal standard solution per dish by immersing the tip of the pipette into the methanol and gently pipetting up and down several times.</li>
	<li>Gently rock each culture dish back and forth to cover its entire surface.</li>
	<li>Leave the dishes at room temperature for 30 s.</li>
</ol>
6. Ultrafiltration of cell extracts
<ol>
	<li>Transfer the extracted solution from each culture dish to a separate 1.5 mL microcentrifuge tube.</li>
	<li>Centrifuge the tubes at 2,300 &#215; g at 4 &#176;C for 5 min.</li>
	<li>Transfer 350 &#956;L of each supernatant into two centrifugal filter units per sample. 
	NOTE: From each culture dish, a total of 700 &#956;L of the extracted solution is transferred into two filter tubes (350 &#956;L/tube).</li>
	<li>Centrifuge the filter tubes at 9,100 &#215; g at 4 &#176;C for approximately 2 h until no liquid remains in the filter cups.</li>
	<li>Remove the filter cups and tightly close the lids of the collection tubes.</li>
</ol>
7. Sample evaporation
<ol>
	<li>Prepare a centrifugal evaporator&#8212;typically, this consists of an evaporator, a cold trap, and a vacuum pump.</li>
	<li>Place the collection tubes in the centrifugal evaporator. 
	NOTE: Leave the lids of the tubes open.</li>
	<li>Evaporate the extracted sample solutions under vacuum conditions at room temperature. 
	NOTE: Typical configurations for the number of rotations and pressure are 1,500 rpm and 1,000 Pa, respectively, and it usually takes approximately 3 h to completely evaporate the samples.</li>
	<li>Confirm that no liquid remains in any of the collection tubes and close the lids of the tubes tightly.</li>
	<li>Store the collection tubes in an ultra-low temperature (&#8722;80 &#176;C) deep freezer until metabolomic analysis.</li>
</ol>
8. Metabolomic analysis by CE-MS
<ol>
	<li>Resuspend the filtrate in 50 &#956;L of ultrapure water immediately before CE-MS analysis.</li>
	<li>Perform CE-MS analysis by methods described previously12,13 using capillary electrophoresis system and time-of-flight mass spectrometer system equipped with an isocratic pump, a CE-MS adapter, and a CE-ESI-MS sprayer. 
	NOTE: Both systems can be controlled by the software of the system vendors and are connected by a fused silica capillary (50 &#956;m internal diameter &#215; 80 cm total length).
	<ol>
		<li>Set up instruments and sample vials, prepare the capillary with a capillary cassette, replenish sheath liquids and appropriate electrophoresis buffers depending on anion or cation analysis mode, and then apply voltages. 
		NOTE: Instrumentation and analytical conditions are described in detail elsewhere12,13.</li>
		<li>Open the software and prepare a worklist containing data acquisition methods and sample information.</li>
		<li>Start a test run and check the data such as the signal intensity and the peak shape of internal standards and the peak resolution of other standard compounds.</li>
		<li>Fine-tune the analytical conditions if necessary.</li>
		<li>Inject the sample solutions at 50 mbar for 3 s and a voltage of 30 kV. 
		NOTE: CE-MS was conducted either in the positive or negative ion mode. Set the spectrometer to scan the mass range m/z 50&#8211;1000. The capillary voltage was set at 4 kV; the flow rate of nitrogen gas (heater temperature 300&#176;C). For positive mode, the fragmentor, skimmer and OCT RFV voltage were set at 75, 50 and 125 V, respectively. For negative ion mode, the fragmentor, skimmer, and OCT RFV voltage was set at 100, 50, and 200 V, respectively.</li>
	</ol>
	</li>
	<li>Analyze spectrum data.
	<ol>
		<li>Extract peaks from the mass spectral data using automatic integration software in order to obtain peak information including m/z, peak area, and migration time (MT). 
		NOTE: The method is described in detail elsewhere14.</li>
		<li>Exclude signal peaks corresponding to isotopomers, adduct ions, and other product ions of known metabolites.</li>
		<li>Annotate remaining peaks with information from the HMT metabolite database according to m/z values and MTs.</li>
		<li>Normalize areas of the annotated peaks to internal standard levels and numbers of cells per sample.</li>
		<li>Evaluate the concentration of each metabolite in the cultured cells (pmol/106 cells) using standard curves prepared for each metabolite.</li>
		<li>Use the quantified metabolite concentrations for subsequent statistical analyses and biological interpretations14.</li>
	</ol>
	</li>
</ol>

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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Warburg+revisited:+imaging+tumour+blood+flow+and+metabolism.">Warburg revisited: imaging tumour blood flow and metabolism.</a> Cancer Imaging. 8, 81-86 (2008).</li><li>Hanahan, D., Weinberg, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hallmarks+of+cancer:+the+next+generation.">Hallmarks of cancer: the next generation.</a> Cell. 144 (5), 646-674 (2011).</li><li>Levine, A. J., Puzio-Kuter, A. 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The authors have nothing to disclose.

Here, we describe a widely accessible methodology to prepare metabolites from cultured cancer cells for CE-MS-based metabolomic analysis. One of the most critical points in this protocol is the proper preparation of cancer cells, because measured metabolite concentrations are normalized to the number of viable cells. For accurate estimation of cell number, it is necessary to prepare at least one additional culture dish per experimental group to count the number of viable cells in parallel with the extraction of metabolites for metabolomic analysis. In addition, the same number of cells should be seeded in each dish for the replicates and in the dish for counting; in the future, this would be aided by a quick and stressor-free (e.g., trypsin-free) cell counting protocol that allows the same dish to be used for both counting viable cells and extracting metabolites. Care should be taken during washes so that cells do not detach from the surface of the dishes. Severe cytotoxicity tests and other experiments that reduce cell adhesion may be unsuitable for this extraction protocol due to potential loss of cells during the washing procedure.
It is important to use a 5% mannitol solution as the wash buffer for extracting metabolites from cultured cells for CE-MS-based metabolomic analysis, because salt-based buffers, such as PBS, interfere with metabolomic analysis and adversely affect measurement.
Two or three dishes can be combined as a single sample by individually extracting metabolites from each dish and then pooling samples; however, combining multiple dishes often increases residual mannitol in the extracted metabolite solution. This may also interfere with metabolomic analysis by CE-MS. Hence, it is recommended to not use multiple dishes or wells as a single sample.
This metabolomic analysis method using CE-MS has been developed for comprehensive measurement of charged molecules with molecular weights between 50 and 1000 Da; thus, this protocol is optimized for extraction of aqueous, low molecular weight compounds. Therefore, this protocol is not suitable for extracting hydrophobic metabolites such as lipids or macromolecules such as proteins and nucleic acids. Since there is an increasing demand for comprehensive lipid analyses or lipidomics of cultured cell samples, the development of an easy and effective protocol for simultaneous extraction of both hydrophilic and hydrophobic metabolites is needed.
The first step of metabolite extraction&#8212;aspirating medium and washing cells with mannitol&#8212;should be conducted as quickly as possible to minimize changes to the metabolic profile of the cells. Treatment of cells with methanol after washing with mannitol is assumed to denature proteins and thereby prevent enzymes from catalyzing further metabolic reactions. However, even after methanol treatment, non-enzymatic chemical reactions&#8212;such as redox reactions, some decarboxylation processes, and thiol linkages&#8212;may take place. As such, any concentrations of metabolites involved in these reactions measured by this protocol should be interpreted with caution. In contrast to the genome or transcriptome, the metabolome consists of molecules with a wide variety of chemical properties; hence, no single protocol can extract all metabolites without any loss or disturbance. For more accurate measurements of such highly reactive metabolites, a protocol specifically designed to extract certain groups of metabolites, which requires fractionations and derivatizations, should be consulted. The protocol presented here, however, describes a simple and quick extraction of aqueous metabolites from cultured cell samples for metabolomic analysis by CE-MS. In this paper we could not describe how to set up CE-MS in detail because the focus of the present manuscript is different, however, describing detailed steps to set up CE-MS may require a separate dedicated article.

Otto Warburg observed that cancer cells acquire the unusual ability to take up glucose and ferment it to produce lactate in the presence of adequate oxygen&#8212;a phenomenon termed as Warburg effect or aerobic glycolysis1,2. Mitochondrial respiration defects are speculated as the underlying basis for aerobic glycolysis in cancer cells3. Indeed, the Warburg effect is the basis for tumor imaging by fluorodeoxyglucose (FDG)-positron emission tomography (PET), which is widely used in clinical practice4,5. A high rate of aerobic glycolysis is considered a key feature of cancer and has been recently adopted as one of the well-known &#8220;hallmarks of cancer,&#8221; as described by D. Hanahan and B. Weinberg6. Somatic mutations in oncogenes and tumor suppressor genes&#8212;such as HRAS/KRAS/NRAS, EGFR, BRAF, MYC, TP53, isocitrate dehydrogenase (IDH), and fumarate hydratase (FH)&#8212;have been linked to specific metabolic changes in cancer cells, believed to be a result of the Warburg effect7.
Metabolomic analysis is a promising approach not only to understand metabolic regulation in cancer cells but also to identify early-stage cancer biomarkers and chemotherapy response prediction. Following treatment of sensitive or resistant cancer cells with anticancer compounds, tracking of their metabolic responses facilitates identification of metabolic biomarkers to predict efficacy of specific anticancer therapies in cancer patients8,9,10,11. In this article, cancer cell lines derived from a lung adenocarcinoma with an EGFR mutation treated with diamide&#8212;which causes oxidative stress&#8212;were used as models for metabolomic analysis. The advantage of this analytical method using capillary electrophoresis-mass spectrometry (CE-MS) is its comprehensive measurement of charged metabolites with the mass range m/z 50-100012,13. The purpose of this article is to provide novices a detailed stepwise visual protocol for preparation of aqueous metabolites from cultured cancer cells and subsequent metabolomic analysis, particularly by CE-MS.

<table><tbody><tr><td>Automated cell counter</td><td>Thermo Scientific</td><td>AMQAX1000</td><td>Countess II automated cell counter</td></tr><tr><td>Automatic integration software</td><td>Agilent Technologies</td><td>MassHunter G3335-60041</td><td>version B.02.00</td></tr><tr><td>CE system</td><td>Agilent Technologies</td><td>Agilent 7100 CE system</td><td></td></tr><tr><td>CE/MS adapter kit</td><td>Agilent Technologies</td><td>G1603A</td><td></td></tr><tr><td>CE-ESI-MS Sprayer kit</td><td>Agilent Technologies</td><td>G1607A</td><td></td></tr><tr><td>Cell counting chamber slide</td><td>Thermo Scientific</td><td>C10282</td><td>Countess cell counting chamber slides</td></tr><tr><td>Centrifugal filter device, 5 kDa</td><td>Human Metabolome Technologies</td><td>ULTRAFREE MC PLHCC, UFC3LCCNB-HMT</td><td></td></tr><tr><td>Conical sterile polypropylene tube, 15 ml</td><td>Thermo Scientific</td><td>N339651</td><td></td></tr><tr><td>Conical sterile polypropylene tube, 50 ml</td><td>Thermo Scientific</td><td>N339653</td><td></td></tr><tr><td>Costar stripette, 10 ml</td><td>Corning</td><td>4488</td><td></td></tr><tr><td>Costar stripette, 5 ml</td><td>Corning</td><td>4487</td><td></td></tr><tr><td>D(-)-Mannitol</td><td>Wako</td><td>133-00845</td><td>500 g</td></tr><tr><td>Dulbecco&#039;s phosphate buffered saline (DPBS)</td><td>Sigma-Aldrich</td><td>D8537-500ML</td><td></td></tr><tr><td>Electrophoresis buffer</td><td>Human Metabolome Technologies</td><td>H3301-1001</td><td>for cation analysis</td></tr><tr><td>Electrophoresis buffer</td><td>Human Metabolome Technologies</td><td>H3302-1021</td><td>for anion analysis</td></tr><tr><td>Fetal bovine serum</td><td>Biowest</td><td>S1780</td><td></td></tr><tr><td>Filter tip, 1000 &amp;mu;l</td><td>Watson</td><td>124P-1000S</td><td></td></tr><tr><td>Filter tip, 20 &amp;mu;l</td><td>Watson</td><td>124P-20S</td><td></td></tr><tr><td>Filter tip, 200 &amp;mu;l</td><td>Watson</td><td>1252-703CS</td><td></td></tr><tr><td>Fused silica capillary</td><td>Polymicro Technologies</td><td>TSP050375</td><td>50 &amp;mu;m i.d. &amp;times; 80 cm total length</td></tr><tr><td>HCC827</td><td>American Type Culture Collection</td><td>CRL-2868</td><td></td></tr><tr><td>Internal standard solution</td><td>Human Metabolome Technologies</td><td>H3304-1002</td><td></td></tr><tr><td>Isocratic pump</td><td>Agilent Technologies</td><td>Agilent 1100 Series Isocratic Pump</td><td></td></tr><tr><td>Methanol</td><td>Wako</td><td>138-14521</td><td>1 L, LC/MS grade</td></tr><tr><td>Microtube, 1.5 ml</td><td>Watson</td><td>131-415C</td><td></td></tr><tr><td>Operating Software</td><td>Agilent Technologies</td><td>ChemStation G2201AA</td><td>version B.03.01 for CE</td></tr><tr><td>PC-9</td><td>RIKEN Bio Resource Center</td><td>RCB4455</td><td></td></tr><tr><td>RPMI-1640</td><td>Sigma-Aldrich</td><td>R8758-500ML</td><td></td></tr><tr><td>Sterile tissue culture dish, 100 mm</td><td>Corning</td><td>430167</td><td></td></tr><tr><td>Time-of-flight mass spectrometer</td><td>Agilent Technologies</td><td>Agilent G1969A Time-of-Flight LC/MS</td><td></td></tr><tr><td>Trypan blue solution, 0.4%</td><td>Thermo Scientific</td><td>T10282</td><td></td></tr><tr><td>Trypsin-EDTA solution</td><td>Sigma-Aldrich</td><td>T4049-100ML</td><td></td></tr><tr><td>Ultrapure water</td><td>Merck</td><td>Milli-Q water</td><td>18.2 M&amp;Omega;&amp;#183;cm pure water</td></tr><tr><td>Volumetric flask, 50 ml</td><td>Iwaki</td><td>5640FK50E</td><td>TE-32</td></tr></tbody></table>

extraction of aqueous metabolites from cultured adherent cells for metabolomic analysis by capillary electrophoresis-mass spectrometry

Metabolomic analysis is a promising omics approach to not only understand the specific metabolic regulation in cancer cells compared to normal cells but also to identify biomarkers for early-stage cancer detection and prediction of chemotherapy response in cancer patients. Preparation of uniform samples for metabolomic analysis is a critical issue that remains to be addressed. Here, we present an easy and reliable protocol for extracting aqueous metabolites from cultured adherent cells for metabolomic analysis using capillary electrophoresis-mass spectrometry (CE-MS). Aqueous metabolites from cultured cells are analyzed by culturing and washing cells, treating cells with methanol, extracting metabolites, and removing proteins and macromolecules with spin columns for CE-MS analysis. Representative results using lung cancer cell lines treated with diamide, an oxidative reagent, illustrate the clearly observable metabolic shift of cells under oxidative stress. This article would be especially valuable to students and investigators involved in metabolomics research, who are new to harvesting metabolites from cell lines for analysis by CE-MS.

Since metabolite concentrations in cancer cells (pmol/106 cells) are normalized to the number of viable cells, experimental conditions should be set up with care so as to minimize variation in the number of viable cells between conditions. For example, diamide treatment was at a relatively high concentration (250 &#956;m) but for a short time to allow all the cells to grow as equally as possible, thereby equalizing the number of viable cells analyzed. Under these experimental conditions, HCC827 and PC-9 cells grew equally for 3 h (Figure 1). CE-MS analysis of diamide-treated cells compared with PBS-treated (control) cells revealed 175 and 150 differential metabolites in HCC827 and PC-9 cells, respectively. Among these, several intermediates in the pentose phosphate pathway (PPP) and in upper glycolysis were significantly higher in the diamide-treated conditions in both cell lines, whereas a few tricarboxylic acid (TCA) cycle intermediates were lower in the treated conditions (Figure 2 and Figure 3).
The PPP generates reducing equivalents in the form of reduced nicotinamide adenine dinucleotide phosphate (NADPH), which is used for redox homeostasis maintenance and fatty acid biosynthesis15. Following diamide treatment, the level of gluconic acid&#8212;an oxidized glucose&#8212;increased 12-fold in HCC827 cells and 10-fold in PC-9 cells; similarly, following diamide treatment, the level of glucose 6-phosphate (G6P)&#8212;a phosphorylated glucose and the first hexokinase-catalyzed glycolysis product&#8212;also increased 6.3- and 3.5-fold in HCC827 and PC-9 cells, respectively (Figure 4). In addition, following diamide treatment, the levels of 6-phosphogluconate (6PG)&#8212;the first intermediate in PPP&#8212;dramatically increased 89-fold in HCC827 cells and 231-fold in PC-9 cells compared to the levels seen in the PBS controls (Figure 4). In contrast, levels of other glycolytic intermediates, such as fructose 6-phosphate (F6P) and fructose 1,6-bisphosphate (F1,6P), did not change in the diamide experimental condition (Figure 4). Total nicotinamide adenine dinucleotide phosphate (NADP+) levels were nearly equivalent between diamide treatment and PBS control conditions (Figure 4), suggesting that glucose was mainly catabolized via the PPP.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59551/59551fig1.jpg" /> 
Figure 1. Unchanged cell numbers upon diamide treatment. Cell growth responses to 250 &#956;m of diamide were measured using trypan blue staining. Cell numbers of (A) HCC827 and (B) PC-9 cells treated with PBS (blue) or diamide (red; 250 &#956;m) for 1 or 3 h are shown. Data are shown as the mean &#177; SD (n = 6). <a href="https://www.jove.com/files/ftp_upload/59551/59551fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59551/59551fig2.jpg" /> 
Figure 2. Representative MS peaks of metabolites. Electropherograms annotated as (A) gluconic acid, (B) glucose 6-phosphate (G6P), (C) 6-phosphogluconate (6PG), and (D) nicotinamide adenine dinucleotide phosphate (NADP+) obtained by CE-MS analysis. Each line indicates the cell line (solid, HCC827; dotted, PC-9) and treatment (blue, PBS; red, diamide) used. <a href="https://www.jove.com/files/ftp_upload/59551/59551fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59551/59551fig3.jpg" /> 
Figure 3. Metabolome profiles of intracellular metabolites. Fold changes of metabolites in (A) HCC827 and (B) PC-9 cells treated with diamide are shown as log2(diamide/PBS). In total, 175 and 150 metabolites were annotated in HCC827 and PC-9 cells, respectively. <a href="https://www.jove.com/files/ftp_upload/59551/59551fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59551/59551fig4.jpg" /> 
Figure 4. Up-regulation of PPP upon diamide treatment. Intracellular concentrations (pmol/106 cells) of key metabolites involved in glycolysis and the pentose phosphate pathway (PPP) after treatment with diamide are shown. Metabolites were extracted from HCC827 and PC-9 cells treated with PBS (blue) or diamide (red, 250 &#956;m) for 30 min. Representative metabolites such as gluconic acid, glucose 6-phosphate (G6P), fructose 6-phosphate (F6P), 6-phosphogluconate (6PG), and nicotinamide adenine dinucleotide phosphate (NADP+) are shown. <a href="https://www.jove.com/files/ftp_upload/59551/59551fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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Extraction of Aqueous Metabolites from Cultured Adherent Cells for Metabolomic Analysis by Capillary Electrophoresis-Mass Spectrometry

The purpose of this article is to describe a protocol for extraction of aqueous metabolites from cultured adherent cells for metabolomic analysis, particularly, capillary electrophoresis-mass spectrometry.

Metabolomic analysis is a promising omics approach to not only understand the specific metabolic regulation in cancer cells compared to normal cells but ...

extraction-aqueous-metabolites-from-cultured-adherent-cells-for

Research

JoVE Journal

Cancer Research

9.0K Views.  Shonai Regional Industry Promotion Center. Het doel van dit artikel is om een gedetailleerd, stapsgewijs visueel protocol te bieden voor het extraheren van waterige metabolieten uit gekweekte kankercellen voor latere metabokoomanalyse. Het belangrijkste voordeel van deze techniek is de eenvoud en betrouwbaarheid voor het extraheren van metabolieten uit aanhangende cellen. Maar bovenal is het goed geoptimaliseerd voor metabolome-analyse met capillaire elektroforese-massaspectrometrie of CEMS.Metabolomics is een krachtige technie...

Video: Extraction of Aqueous Metabolites from Cultured Adherent Cells for Metabolomic Analysis by Capillary Electrophoresis-Mass Spectrometry

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 ...

Chemistry

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

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

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

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

Biochemistry

A Method for Measuring Metabolism in Sorted Subpopulations of Complex Cell Communities Using Stable Isotope Tracing

Mammalian cell types exhibit specialized metabolism, and there is ample evidence that various co-existing cell types engage in metabolic cooperation. Moreover, even cultures of a single cell type may contain cells in distinct metabolic states, such as resting or cycling cells. Methods for measuring metabolic activities of such subpopulations are valuable tools for understanding cellular metabolism. Complex cell populations are most commonly separated using a cell sorter, and subpopulations isolated by this method can be analyzed by metabolomics methods. However, a problem with this approach is that the cell sorting procedure subjects cells to stresses that may distort their metabolism.
To overcome these issues, we reasoned that the mass isotopomer distributions (MIDs) of metabolites from cells cultured with stable isotope-labeled nutrients are likely to be more stable than absolute metabolite concentrations, because MIDs are formed over longer time scales and should be less affected by short-term exposure to cell sorting conditions. Here, we describe a method based on this principle, combining cell sorting with liquid chromatography-high resolution mass spectrometry (LC-HRMS). The procedure involves analyzing three types of samples: (1) metabolite extracts obtained directly from the complex population; (2) extracts of "mock sorted" cells passed through the cell sorter instrument without gating any specific population; and (3) extracts of the actual sorted populations. The mock sorted cells are compared against direct extraction to verify that MIDs are indeed not altered by the cell sorting procedure itself, prior to analyzing the actual sorted populations. We show example results from HeLa cells sorted according to cell cycle phase, revealing changes in nucleotide metabolism.

This article describes a method for studying cellular metabolism in complex communities of multiple cell types, using a combination of stable isotope tracing, cell sorting to isolate specific cell types, and mass spectrometry.

Mammalian cell types exhibit specialized metabolism, and there is ample evidence that various co-existing cell types engage in metabolic cooperation. ...

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

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

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

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

Microprobe Capillary Electrophoresis Mass Spectrometry for Single-cell Metabolomics in Live Frog (Xenopus laevis) Embryos

The quantification of small molecules in single cells raises new potentials for better understanding the basic processes that underlie embryonic development. To enable single-cell investigations directly in live embryos, new analytical approaches are needed, particularly those that are sensitive, selective, quantitative, robust, and scalable to different cell sizes. Here, we present a protocol that enables the in situ analysis of metabolism in single cells in freely developing embryos of the South African clawed frog (Xenopus laevis), a powerful model in cell and developmental biology. This approach uses a capillary microprobe to aspirate a defined portion from single identified cells in the embryo, leaving neighboring cells intact for subsequent analysis. The collected cell content is analyzed by a microscale capillary electrophoresis electrospray ionization (CE-ESI) interface coupled to a high-resolution tandem mass spectrometer. This approach is scalable to various cell sizes and compatible with the complex three-dimensional structure of the developing embryo. As an example, we demonstrate that microprobe single-cell CE-ESI-MS enables the elucidation of metabolic cell heterogeneity that unfolds as a progenitor cell gives rise to descendants during development of the embryo. Besides cell and developmental biology, the single-cell analysis protocols described here are amenable to other cell sizes, cell types, or animal models.

Microprobe Capillary Electrophoresis Mass Spectrometry for Single-cell Metabolomics in Live Frog Xenopus laevis Embryos

We describe steps that enable fast in situ sampling of a small portion of an individual cell with high precision and minimal invasion using capillary-based micro-sampling, to facilitate chemical characterization of a snapshot of metabolic activity in live embryos using a custom-built single cell capillary electrophoresis and mass spectrometry platform.

The quantification of small molecules in single cells raises new potentials for better understanding the basic processes that underlie embryonic ...

Multi-step Preparation Technique to Recover Multiple Metabolite Compound Classes for In-depth and Informative Metabolomic Analysis

Metabolomics is an emerging field which enables profiling of samples from living organisms in order to obtain insight into biological processes. A vital aspect of metabolomics is sample preparation whereby inconsistent techniques generate unreliable results. This technique encompasses protein precipitation, liquid-liquid extraction, and solid-phase extraction as a means of fractionating metabolites into four distinct classes.&#160;Improved enrichment of low abundance molecules with a resulting increase in sensitivity is obtained, and ultimately results in more confident identification of molecules. This technique has been applied to plasma, bronchoalveolar lavage fluid, and cerebrospinal fluid samples with volumes as low as 50 &#181;l. &#160;Samples can be used for multiple downstream applications; for example, the pellet resulting from protein precipitation can be stored for later analysis. The supernatant from that step undergoes liquid-liquid extraction using water and strong organic solvent to separate the hydrophilic and hydrophobic compounds. Once fractionated, the hydrophilic layer can be processed for later analysis or discarded if not needed. The hydrophobic fraction is further treated with a series of solvents during three solid-phase extraction steps to separate it into fatty acids, neutral lipids, and phospholipids. This allows the technician the flexibility to choose which class of compounds is preferred for analysis. It also aids in more reliable metabolite identification since some knowledge of chemical class exists.

The reliability of results in metabolomics experiments depends on the effectiveness and reproducibility of the sample preparation. Described is a rigorous and in-depth method that enables extraction of metabolites from biological fluids with the option of subsequently analyzing up to thousands of compounds, or just the compound classes of interest.

Metabolomics is an emerging field which enables profiling of samples from living organisms in order to obtain insight into biological processes. A vital ...

Bioengineering

Absolute Quantification of Cell-Free Protein Synthesis Metabolism by Reversed-Phase Liquid Chromatography-Mass Spectrometry

Cell-free protein synthesis (CFPS) is an emerging technology in systems and synthetic biology for the in vitro production of proteins. However, if CFPS is going to move beyond the laboratory and become a widespread and standard just in time manufacturing technology, we must understand the performance limits of these systems. Toward this question, we developed a robust protocol to quantify 40 compounds involved in glycolysis, the pentose phosphate pathway, the tricarboxylic acid cycle, energy metabolism and cofactor regeneration in CFPS reactions. The method uses internal standards tagged with 13C-aniline, while compounds in the sample are derivatized with 12C-aniline. The internal standards and sample were mixed and analyzed by reversed-phase liquid chromatography-mass spectrometry (LC/MS). The co-elution of compounds eliminated ion suppression, allowing the accurate quantification of metabolite concentrations over 2-3 orders of magnitude where the average correlation coefficient was 0.988. Five of the forty compounds were untagged with aniline, however, they were still detected in the CFPS sample and quantified with a standard curve method. The chromatographic run takes approximately 10 min to complete. Taken together, we developed a fast, robust method to separate and accurately quantify 40 compounds involved in CFPS in a single LC/MS run. The method is a comprehensive and accurate approach to characterize cell-free metabolism, so that ultimately, we can understand and improve the yield, productivity and energy efficiency of cell-free systems.

Here, we present a robust protocol to quantify 40 compounds involved in central carbon and energy metabolism in cell-free protein synthesis reactions. The cell-free synthesis mixture is derivatized with aniline for effective separation using reversed-phase liquid chromatography and then quantified by mass spectrometry using isotopically labelled internal standards.

Cell-free protein synthesis (CFPS) is an emerging technology in systems and synthetic biology for the in vitro production of proteins. However, if CFPS is ...

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

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

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

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

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

Exploring Metabolite Exchange in Brown Adipose Tissue

Arteriovenous Metabolomics to Measure In Vivo Metabolite Exchange in Brown Adipose Tissue

Brown adipose tissue (BAT) plays a crucial role in regulating metabolic homeostasis through a unique energy expenditure process known as non-shivering thermogenesis. To achieve this, BAT utilizes a diverse menu of circulating nutrients to support its high metabolic demand. Additionally, BAT secretes metabolite-derived bioactive factors that can serve as either metabolic fuels or signaling molecules, facilitating BAT-mediated intratissue and/or intertissue communication. This suggests that BAT actively participates in systemic metabolite exchange, an interesting feature that is beginning to be explored. Here, we introduce a protocol for in vivo mouse-level optimized BAT arteriovenous metabolomics. The protocol focuses on relevant methods for thermogenic stimulations and an arteriovenous blood sampling technique using Sulzer's vein, which selectively drains interscapular BAT-derived venous blood and systemic arterial blood. Next, a gas chromatography-based metabolomics protocol using those blood samples is demonstrated. The use of this technique should expand the understanding of BAT-regulated metabolite exchange at the inter-organ level by measuring the net uptake and release of metabolites by BAT.

Author Spotlight: Illuminating New Avenues for Adipose Tissue Metabolism and Disease Prevention 

In this protocol, methods relevant for BAT-optimized arteriovenous metabolomics using GC-MS in a mouse model are outlined. These methods allow for the acquisition of valuable insights into BAT-mediated metabolite exchange at the organismal level.

Brown adipose tissue (BAT) plays a crucial role in regulating metabolic homeostasis through a unique energy expenditure process known as non-shivering ...

Liquid Chromatography Coupled to Refractive Index or Mass Spectrometric Detection for Metabolite Profiling in Lysate-based Cell-free Systems

Engineering cellular metabolism for targeted biosynthesis can require extensive design-build-test-learn (DBTL) cycles as the engineer works around the cell&#39;s survival requirements. Alternatively, carrying out DBTL cycles in cell-free environments can accelerate this process and alleviate concerns with host compatibility. A promising approach to cell-free metabolic engineering (CFME) leverages metabolically active crude cell extracts as platforms for biomanufacturing and for rapidly discovering and prototyping modified proteins and pathways. Realizing these capabilities and optimizing CFME performance requires methods to characterize the metabolome of lysate-based cell-free platforms. That is, analytical tools are necessary for monitoring improvements in targeted metabolite conversions and in elucidating alterations to metabolite flux when manipulating lysate metabolism. Here, metabolite analyses using high-performance liquid chromatography (HPLC) coupled with either optical or mass spectrometric detection were applied to characterize metabolite production and flux in E. coli S30 lysates. Specifically, this report describes the preparation of samples from CFME lysates for HPLC analyses using refractive index detection (RID) to quantify the generation of central metabolic intermediates and by-products in the conversion of low-cost substrates (i.e., glucose) to various high-value products. The analysis of metabolite conversion in CFME reactions fed with 13C-labeled glucose through reversed-phase liquid chromatography coupled to tandem mass spectrometry (MS/MS), a powerful tool for characterizing specific metabolite yields and lysate metabolic flux from starting materials, is also presented. Altogether, applying these analytical methods to CFME lysate metabolism enables the advancement of these systems as alternative platforms for executing faster or novel metabolic engineering tasks.

The protocols describe high-performance liquid chromatography methods coupled to refractive index or mass spectrometric detection for studying metabolic reactions in complex lysate-based cell-free systems.

Engineering cellular metabolism for targeted biosynthesis can require extensive design-build-test-learn (DBTL) cycles as the engineer works around the ...

Extraction of Aqueous Metabolites from Cultured Adherent Cells for Metabolomic Analysis by Capillary Electrophoresis-Mass Spectrometry

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