This work was supported by a research grant from the Ministry of Education, Culture, Sports, Science and Technology of Japan (KAKENHI) to K.H. (#15K01691), Y.F. (#15K08625), K.Y. (#26461532), and a grant for the study of Intractable Disease Project from Ministry of Health, Labour and Welfare (K.Y. #201510032A). We thank the Edanz Group (www.edanzediting.com/ac) for editing a draft of this manuscript.

Consult all relevant material safety data sheets (MSDS) before use. Wear gloves to minimize sample contamination by skin-derived SM. The present protocol was applied to HeLa cells grown in Eagle&#39;s minimum essential medium supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 1,000 U/L penicillin, and 100 mg/L streptomycin.
1. Preparation of Lipid Samples
NOTE: It is important that all glassware including test tubes with Teflon-lined screw caps be detergent-free.
<ol>
	<li>Extraction of total lipid fraction from cell homogenate using the Bligh &#38; Dyer method7
	<ol>
		<li>Remove the culture (or conditioned) media from the 10-cm tissue culture dish and rinse twice with 6 mL of ice-cold PBS.</li>
		<li>Add 1 mL of ice-cold PBS into the 10-cm tissue culture dish by P1000 pipette. Harvest the cells with cell scrapers and collect into 2.0-mL siliconized plastic tubes.</li>
		<li>After centrifugation (1,000 &#215; g, 5 min, 4 &#176;C), remove the supernatant by a P1000 pipette.</li>
		<li>Add 1 mL of methanol. Vortex tubes briefly and sonicate at 200 W for 5 min in a bath-type sonicator. 
		NOTE: Adjust the water level in a bath-type sonicator so that the cell pellet is efficiently homogenized.</li>
		<li>Transfer the cell homogenate in methanol by pipette into test tubes (13 mm x 100 mm) with Teflon-lined screw caps.</li>
		<li>Add 1 mL of methanol, 1 mL of chloroform, 0.8 mL of double distilled water, and 50 &#181;L of 10 &#181;mol/L of internal standard d18:1/(D31)-16:0 SM into each test tube.</li>
		<li>Vortex test tubes vigorously for 5 min at room temperature. 
		NOTE: At this point, a single phase (chloroform/methanol/water = 1/2/0.8 (v/v/v)) is formed.</li>
		<li>Add 1 mL of chloroform and 1.0 mL of double distilled water.</li>
		<li>Vortex tubes vigorously at 2,500 rpm for 5 min at room temperature. 
		NOTE: At this point, the aqueous (upper) phase and organic (lower) phase are separated.</li>
		<li>After centrifugation (2,150 &#215; g, 5 min, 25 &#176;C), collect and transfer the lower phase into disposable glass tubes (initial lower phase). 
		&#8203;NOTE: Collect the lower phase using a Pasteur pipette and a safety pipette filter. Insert the tip of the pipette into the lower phase, squeeze the small bulb next to the &#39;E&#39; valve to deliver the upper phase and interfacial fluff inside the tip of the pipette, and then siphon the lower phase into the pipette.</li>
		<li>Add 2 mL of chloroform into the test tubes, and vortex the tubes vigorously at 2,500 rpm for 5 min at room temperature to sufficiently mix with the upper phase and interfacial fluff.</li>
		<li>After centrifugation (2,150 &#215; g, 5 min, 25 &#176;C), collect and transfer the modified lower phase into the disposable glass tubes with the initial lower phase as described in step 1.1.10.</li>
		<li>Place the glass tubes under a nitrogen stream and completely remove the organic solvents in the collected lower phase.</li>
		<li>Reconstitute the samples with 500 &#181;L of methanol or ethanol, filtrate with a 0.02-&#181;m filter, and store in glass vials at -20 &#176;C.</li>
	</ol>
	</li>
	<li>Sample preparation for constructing the calibration curve and validating the method
	<ol>
		<li>Add 50 &#181;L of 0.1, 0.5, 1, 5, 10, or 50 &#181;mol/L of standard compound (d18:1/(D9)-18:1 SM) into test tubes with Teflon-lined screw caps.</li>
		<li>Add cell homogenate into each test tube and add methanol to 1 mL. 
		NOTE: The amount of cell homogenate as a matrix varies according to each experiment. If the SM species in samples derived from cells in a 10-cm culture dish are routinely analyzed, the amount of cell homogenate in each test tube should be close to that of cells in a 10-cm culture dish.</li>
		<li>Extract the total lipid fraction as described in steps 1.1.6-1.1.14.</li>
		<li>Prepare quality check (QC) samples for validating the method as descried in steps 1.2.1-1.2.3. Prepare three QC samples with different concentrations of standard compound (d18:1/(D9)-18:1 SM): one within 3x the lower boundary of the standard curve (QC-low, QC-L), one near the center (QC-middle, QC-M), and one near the upper boundary of the standard curve (QC-high, QC-H).</li>
	</ol>
	</li>
</ol>
2. SM Analysis by LC-ESI-MS/MS
<ol>
	<li>Preparation of the mobile phase
	<ol>
		<li>Mix the solvents (acetonitrile/methanol/ddH2O = 2/2/1 (v/v/v) for the aqueous phase and isopropanol for the organic phase) in glass bottles with Teflon-lined screw caps and sonicate for 5 min in a bath-type sonicator.</li>
		<li>Add formic acid (final concentration 26.4 mmol/L) and NH4OH (14.9 mmol/L) into each mobile phase.</li>
	</ol>
	</li>
	<li>Qualitative analysis of SM by LC-ESI-MS3
	<ol>
		<li>Activate the high performance liquid chromatography (HPLC) system, put inlet tubes into the glass bottles that contain mobile phases, and purge the HPLC lines. Link a C18 HPLC column (1.5 mm i.d. &#215; 100 mm length, particle size 3.0 &#181;m) to the HPLC system, keep the temperature in the column oven at 50 &#176;C, and condition the column with the aqueous mobile phase at 100 &#181;L/min. Put the samples into a sample rack in the autosampler.</li>
		<li>Set the parameters of the triple quadrupole and quadrupole linear ion trap mass spectrometry system for MS3 analysis as listed in Table 1 and Table 2 as well as the first and second precursor ions of the SM species of interest.
		<ol>
			<li>Double click the software icon for data acquisition, double click the &#39;Hardware Configuration&#39;, select &#39;LC+QTRAP4500+Valve&#39;, and click &#39;Activate Profile&#39;.</li>
			<li>Create a new subfolder by clicking &#39;New Sub-Project&#39; and &#39;OK&#39;.</li>
			<li>Click the &#39;file&#39; tab, select &#39;New&#39;, and select &#39;Acquisition Method&#39; and &#39;OK&#39;. Click the &#39;Mass Spec&#39; icon and &#39;MS&#39; tab. Select Scan type as &#39;MS/MS/MS (MS3)&#39;, Scan rate as 10,000 Da/s, Polarity as &#39;Negative&#39;, and &#39;Duration&#39; as entire time to be analyzed (min). Set the m/z of the &#39;Start (Da)&#39; and &#39;Stop (Da)&#39; as the range to be scanned. Set the m/z of the 1st and 2nd precursor ions of the target SM species of interest. 
			NOTE: To analyze multiple SM species, highlight the &#39;-MS3&#39; icon, right click, select &#39;Copy this experiment&#39;, and then put the m/z of the 1st and 2nd precursor ions of other SM species.</li>
			<li>Select the &#39;Advanced MS&#39; tab, and set each parameter as follows: Scan mode as &#39;Profile&#39;, Step size as 0.12 (Da), Resolution Q1 and Q3 &#39;Unit&#39; and &#39;LIT&#39;, respectively, Intensity threshold as 0, Setting time as 50 (ms), Pause between mass ranges as 1.5 ms, select &#39;Dynamic fill time&#39;, Q3 Entry Barrier as 8 V, and Excitation Time as 25 ms.</li>
			<li>Click the &#39;Edit Parameters&#39; button in the &#39;MS&#39; tab and select the &#39;Source/Gas&#39; tab. Set the parameters of the curtain gas, collision gas, ionspray voltage, temperature, ion source gas1 and gas2 as listed in Table 1. Then select the &#39;Compound&#39; tab. Set the parameters of the declustering potential, entrance potential, collision energy, excitation energy, and collision energy spread as listed in Table 2.</li>
			<li>Highlight &#39;Integrated Valco Valve&#39; and select &#39;Position Name for Step 0&#39; as A, and set &#39;B&#39; as position for total time 5.0 min. Also set &#39;A&#39; as position for total time 70.0 min.</li>
			<li>Highlight &#39;Shimadzu LC system&#39;. Select the &#39;Pump&#39; tab and set Pumping mode as binary flow, total flow as 0.28 mL/min, and maximum of pressure limits as 20.0 MPa. Select the &#39;Autosampler&#39; tab and set the rinsing volume as 200 &#181;L, needle Stroke as 52 mm, rinsing speed as 35 &#181;L/s, sampling Speed as 5.0 &#181;L/s, purge time as 25.0 min, rinse dip time as 5 s, and rinse mode as &#39;Before and after aspiration&#39;.
			<ol>
				<li>In addition, enable the cooler unit, set the cooler temperature as 4 &#176;C and the control vial needle stroke as 52 mm. Select the &#39;Oven&#39; tab, enable the oven and set the oven temperature and maximum temperature as 50 &#176;C and 85 &#176;C, respectively.</li>
				<li>Select the &#39;Controller&#39; tab and check the box &#39;Power on&#39;. Select the &#39;Time Program&#39; tab and set the solvent gradients as follows: Solvents A/B at a 100/0 ratio for 5 min, program linear alterations to 80/20 over 4 min, to 35/65 over 50 min, and to 25/75 over 1 min. Afterwards, hold them at 25/75 for 10 min, then linearly to 100/0 over 4 min. Then save the method.</li>
			</ol>
			</li>
		</ol>
		</li>
		<li>Create batch file
		<ol>
			<li>Double click the &#39;Build Acquisition Batch&#39; icon, click &#39;Add Set&#39;, &#39;Add Samples&#39;, set the number of new samples, and click &#39;OK&#39;.</li>
			<li>Click the &#39;Method Editor&#39; button and select the method to be used. If multiple methods are used in one batch file, check the box of &#39;Use Multiple Methods&#39; and select the acquisition method for each sample. Rename &#39;Sample Name&#39;, set the appropriate number for &#39;Vial Position&#39;, and put the amount of injection volume. Then save the batch file.</li>
		</ol>
		</li>
		<li>Obtain the MS3 product ion spectra of the SM species of interest by LC-ESI-MS3 analysis
		<ol>
			<li>Select the &#39;Submit&#39; tab in batch file, highlight the line of samples to be analyzed, and click the &#39;Submit&#39; button.</li>
			<li>Click the &#39;&#39;View Quene&#39; icon, &#39;Equilibrate&#39; icon, select the acquisition method to be used, set Time as 1 min, and click &#39;OK&#39;.</li>
			<li>Deactivate &#39;Reserve Instrument for Tuning&#39; by clicking the corresponding icon. Then execute batch sequence by clicking the &#39;Start Sample&#39; icon.</li>
		</ol>
		</li>
		<li>Assign each MS3 product ion spectra of the SM by comparing the mass to charge ratio (m/z) of the product ion spectra and the exact mass of the sphingoid LCB and N-acyl moiety of interest. Determine the number of carbons and double bonds in the sphingoid LCB and N-acyl moiety according to the corresponding product ions.
		<ol>
			<li>Confirm that the appropriate sub-project folder is selected, then double click the &#39;Open Data File&#39; icon, and select the samples to be analyzed. Drag the peak in chromatogram for each target SM species and then double click. The obtained MS3 product ion spectra within the dragged area will be presented.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Quantitative analysis of the SM by LC-ESI-MS/MS
	<ol>
		<li>Set the mobile phases and HPLC column as described in step 2.1 and 2.2.1.</li>
		<li>Set the parameters of the triple quadrupole and quadrupole linear ion trap mass spectrometry system for multiple reaction monitoring (MRM) analysis as listed in Table 1 and Table 2.
		<ol>
			<li>Conduct the procedures as described in step 2.2.2.1 and 2.2.2.2.</li>
			<li>Click the &#39;file&#39; tab, select &#39;New&#39;, and select &#39;Acquisition Method&#39; and &#39;OK&#39;. Click the &#39;Mass Spec&#39; icon and &#39;MS&#39; tab. Select Scan type as &#39;MRM&#39;, Polarity as &#39;Positive&#39;. Set &#39;Duration&#39; as the entire time to be analyzed. Set the m/z of [M+H]+ and 184 as &#39;Q1 Mass (Da)&#39; and &#39;Q3 Mass (Da)&#39; of the target SM species of interest, respectively. Set &#39;Time&#39; as 10 ms and &#39;ID&#39; as the name of the target SM species.</li>
			<li>Select the &#39;Advanced MS&#39; tab, and set each parameter as follows: Both of the resolution Q1 and Q3 as &#39;Unit&#39;, Intensity threshold as 0, Setting time as 0 (ms), and Pause between mass ranges as 5 ms.</li>
			<li>Click the &#39;Edit Parameters&#39; button in the &#39;MS&#39; tab and select the &#39;Source/Gas&#39; tab. Put the parameter of curtain gas, collision gas, ionspray voltage, temperature, ion source gas1 and gas2 as listed in Table 1. Then select the &#39;Compound&#39; tab. Put the parameters of declustering potential, entrance potential, collision energy, and collision cell exit potential as listed in Table 2.</li>
			<li>Set the valve as described in step 2.2.2.6.</li>
			<li>Set the LC condition as described in step 2.2.2.7. Then save the method.</li>
		</ol>
		</li>
		<li>Create the batch file as described in step 2.2.3.</li>
		<li>Obtain the MRM data of each SM species by LC-ESI-MS/MS analysis as described in step 2.2.4.</li>
		<li>Process the MRM data using the software for data integration and obtain the data of peak area for the extracted ion chromatogram of each SM species.
		<ol>
			<li>Double click the software icon for data integration in quantitative analysis, click the &#39;Edit&#39; tab and select User Integration Defaults. Set Gaussian Smooth Width as 1.0 point, and click &#39;OK&#39;. Click the &#39;Edit&#39; tab and select &#39;New Results Table&#39;. Select the sample to be integrated, click an arrow button, and click &#39;Next&#39;. Select &#39;Create New Method&#39;, set the name of the method, and click &#39;Next&#39;. Click &#39;Next&#39; and check the box of d18:1/(D31)-16:0 SM as IS, click &#39;Next&#39;, and click &#39;Finish&#39;.</li>
			<li>Click &#39;Displays the peak review&#39; and confirm that the chromatogram peaks are appropriately recognized. It should be noted that the retention time of d18:1/(D31)-16:0 SM is usually smaller by ~ 0.3 min as compared with the one of the 34-1 SM (usually consists of d18:1/16:0 SM).</li>
		</ol>
		</li>
		<li>Quantify each SM species according to the ratio of the peak of each SM species to the d18:1/(D31)-16:0 SM internal standard.
		<ol>
			<li>Click the &#39;File&#39; tab, select &#39;Export&#39;, select &#39;Results Table&#39;, confirm the format as &#39;MultiQuant&#39;, columns as &#39;Export all columns&#39;, rows as &#39;Export all rows except those explicitly hidden&#39;, and then click &#39;OK&#39;.</li>
			<li>Open the exported file in the Excel software. Normalize the area of the target SM species by the area of IS (d18:1/(D31)-16:0 SM). Then multiply by the theoretical amount of the IS in the injected sample to calculate the amount of target SM species in the injected sample.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Constructing the standard curve
	<ol>
		<li>Obtain the MRM data of d18:1/(D9)-18:1 SM and d18:1/(D31)-16:0 SM in the samples for constructing the standard curve by LC-ESI-MS/MS analysis as described in step 2.3.1-2.3.4.</li>
		<li>Obtain the data of the peak area of d18:1/(D9)-18:1 SM and d18:1/(D31)-16:0 SM as described in step 2.3.5.</li>
		<li>Calculate the amount of d18:1/(D9)-18:1 SM in the injected sample as described in step 2.3.6.</li>
		<li>Construct the trendline by setting the X-axis and Y-axis as the nominal amount and calculated amount of d18:1/(D9)-18:1 SM and obtain the trendline formula.</li>
	</ol>
	</li>
	<li>Validating the quantitative method using Excel software
	<ol>
		<li>For validation of the method, quantify the amount of d18:1/(D9)-18:1 SM and d18:1/(D31)-16:0 SM in the QC samples by analyzing each QC sample at least three times in 1 day, and repeat at least 3 days as described in step 2.3.1-2.3.4.</li>
		<li>Obtain the peak area data and calculate the amount of d18:1/(D9)-18:1 SM in the injected sample as described in step 2.3.5 and 2.3.6.</li>
		<li>According to the calibration curve obtained, calculate the amount of d18:1/(D9)-18:1 SM in the injected sample.</li>
		<li>Evaluation of precision
		<ol>
			<li>Calculate the average and the standard deviation of the amount of d18:1/(D9)-18:1 SM obtained in step 2.5.3 on the dataset of the intra-day and inter-day using Excel software.</li>
			<li>The average obtained in step 2.5.4.1 is divided by the standard deviation, and expressed as %.</li>
		</ol>
		</li>
		<li>Evaluation of accuracy
		<ol>
			<li>Calculate the accuracy of each data as follows: 
			[(The calculated amount obtained in step 2.5.3.) / (Nominal amount) - 1] &#215; 100(%)</li>
			<li>Calculate the average of the absolute value of the accuracy on the dataset of the intra-day and inter-day.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+family+of+animal+sphingomyelin+synthases.">Identification of a family of animal sphingomyelin synthases.</a> EMBO J. 23 (1), 33-44 (2004).</li><li>Kihara, Y., Mizuno, H., Chun, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lysophospholipid+receptors+in+drug+discovery.">Lysophospholipid receptors in drug discovery.</a> Exp Cell Res. 333 (2), 171-177 (2015).</li><li>Kihara, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+and+degradation+pathways,+functions,+and+pathology+of+ceramides+and+epidermal+acylceramides.">Synthesis and degradation pathways, functions, and pathology of ceramides and epidermal acylceramides.</a> Prog Lipid Res. 63, 50-69 (2016).</li><li>Merrill, A. 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The authors declare that they have no conflict of interest.

In the present qualitative method, we obtained MS3 product ions of a SPC and an N-acyl moiety. It is critical to properly assign both a SPC and an N-acyl moiety. To this end, it should be noted that other phosphorylcholine-containing molecules can also be detected as MS3 product ions. Diacyl-phosphatidylcholine (PC) and plasmalogen-PC are abundantly present in mammalian cells, and their hydrophobicity is similar to that of SM. Therefore, diacyl-PC and plasmalogen-PC with an isotope...

Sphingomyelin (SM) is a common sphingolipid in mammalian cells. SM is synthesized intracellularly1 and present as a precursor for other sphingolipids such as a sphingosine-1-phosphate and a ceramide, which have crucial roles in immune cell trafficking and skin barrier homeostasis, respectively2,3. Thus, the precise analysis of the SM metabolism is important for elucidating the physiological and pathological roles of sphingolipids.
SM is composed of a ceramide and a phosphorylcholine that is linked to the 1-hydroxy group of the ceramide, which is further composed of a sphingosine and an N-acyl moiety. A variety in the carbon and double bond numbers in both the sphingosine and N-acyl moiety results in the number of ceramide (and SM) species. Recent advances in LC-ESI-MS/MS has enabled quantitative and qualitative analysis of SM4,5. In the qualitative analysis, the number of carbon and double bonds of a sphingoid LCB of SM was identified by assigning product ion spectra of LCB. However, structural information of the N-acyl moiety was not directly obtained because its corresponding product ions have not been reported, and therefore N-acyl moieties were deduced by differential analysis between precursor ions and product ions corresponding to LCB in both positive and negative ion modes4,5. In this report, we present a method to detect the product ions of both LCB and N-acyl moiety simultaneously in MS3 mode using triple quadrupole and quadrupole linear ion trap mass spectrometry, which facilitates the precise structural speculation of each SM species6.
The ion suppression (or enhancement) effects caused by the matrix in biological samples hamper the accurate quantification in LC-ESI-MS analysis, and therefore, it is desirable to construct calibration curves for all analytes of interest in the identical matrix of the biological sample. However, this strategy is not feasible because it is almost impossible to prepare all SM species in biological samples, especially in comprehensive analysis. Thus, it is practical to construct a calibration curve and determine the quantitative range using a representative SM species spiked in the biological samples. We used two isotopically-labeled SM species to construct a calibration curve; one was used for an internal standard and the other for a standard compound. We detected a small amount of isotopically-labeled SM species as a standard compound spiked in biological samples and successfully obtained a calibration curve and the quantitative range6.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">PBS</td>
			<td style="width:131px;">ThermoFisher</td>
			<td style="width:119px;">10010023</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">100 mm tissue culture dish</td>
			<td style="width:131px;">IWAKI</td>
			<td style="width:119px;">3020-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Cell scraper</td>
			<td style="width:131px;">IWAKI</td>
			<td style="width:119px;">9000-220</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Siliconized 2.0 mL tube</td>
			<td style="width:131px;">Fisher Scientific</td>
			<td style="width:119px;">02-681-321</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Test tube</td>
			<td style="width:131px;">IWAKI</td>
			<td style="width:119px;">TST SCR 16-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Teflon-lined screw cap</td>
			<td style="width:131px;">IWAKI</td>
			<td style="width:119px;">9998CAP415-15</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Disposable glass tube</td>
			<td style="width:131px;">IWAKI</td>
			<td style="width:119px;">9832-1310</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:321px;">CAPCELL PAK C18 ACR 3 &#181;m 1.5 mm I.D. x 100 mm</td>
			<td style="width:131px;">Shiseido</td>
			<td style="width:119px;">92223</td>
			<td>Guard cartridge is inserted into cartridge holder, and linked to C18 column</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:321px;">CAPCELL C18 MGII S-3 2.0 mm x 10 mm GUARD CARTRIDGE</td>
			<td style="width:131px;">Shiseido</td>
			<td style="width:119px;">12197</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Cartridge holder</td>
			<td style="width:131px;">Shiseido</td>
			<td style="width:119px;">12415</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Acetonitrile</td>
			<td style="width:131px;">Wako</td>
			<td style="width:119px;">018-19853</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">2-Propanol (Isopropyl Alcohol)</td>
			<td style="width:131px;">Wako</td>
			<td style="width:119px;">161-09163</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Methanol</td>
			<td style="width:131px;">Wako</td>
			<td style="width:119px;">134-14523</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Formic acid</td>
			<td style="width:131px;">Wako</td>
			<td style="width:119px;">066-00466</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">28% Ammonia water</td>
			<td style="width:131px;">Wako</td>
			<td style="width:119px;">016-03146</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Sonicator (bath type)</td>
			<td style="width:131px;">SHARP</td>
			<td style="width:119px;">UT-206H</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Vortex mixer for glass test tube</td>
			<td style="width:131px;">TAITEC</td>
			<td style="width:119px;">Mix-EVR</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">1.4 mL glass vial</td>
			<td style="width:131px;">Tomsic</td>
			<td style="width:119px;">500-1982</td>
			<td>Samples are stored in 1.4 mL glass vial sealed with screw cap and 8 mm septum at -20&#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">8 mm septum</td>
			<td style="width:131px;">Tomsic</td>
			<td style="width:119px;">200-3322</td>
			<td>When samples are analyzed, screw caps are replaced with screw caps with slit septum</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Screw cap for 1.4 mm glass vial</td>
			<td style="width:131px;">Tomsic</td>
			<td style="width:119px;">500-2762</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Screw cap with slit septum</td>
			<td style="width:131px;">Shimadzu GLC</td>
			<td style="width:119px;">GLCTV-803</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">PVDF 0.22 &#181;m filter</td>
			<td style="width:131px;">Millipore</td>
			<td style="width:119px;">SLGVR04NL</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:321px;">Triple quadrupole and quadrupole linear ion trap mass spectrometry</td>
			<td style="width:131px;">SCIEX</td>
			<td style="width:119px;">QTRAP4500</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:321px;">The software for data acquisition and analysis of product ion spectra</td>
			<td style="width:131px;">SCIEX</td>
			<td style="width:119px;">Analyst</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:321px;">The software for data integration in quantitative analysis</td>
			<td style="width:131px;">SCIEX</td>
			<td style="width:119px;">MultiQuant</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">HPLC system</td>
			<td style="width:131px;">Shimadzu</td>
			<td style="width:119px;">Nexera</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Glass bottle</td>
			<td style="width:131px;">Sansyo</td>
			<td style="width:119px;">85-0002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">d18:1/24:0 sphingomyelin</td>
			<td style="width:131px;">Avanti Polar Lipids</td>
			<td style="width:119px;">860592P</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Sphingosylphosphorylcholine</td>
			<td style="width:131px;">Merck</td>
			<td style="width:119px;">567735</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal bovine serum</td>
			<td style="width:131px;">ThermoFisher&#160;</td>
			<td style="width:119px;">26140079</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">L-glutamine</td>
			<td style="width:131px;">ThermoFisher&#160;</td>
			<td style="width:119px;">25030081</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Penicillin and streptomycin</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:119px;">P4333</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Eagle&#8217;s minimum essential medium</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:119px;">M4655</td>
			<td></td>
		</tr>
	</tbody>
</table>

quantitative and qualitative method for sphingomyelin by lc-ms using two stable isotopically labeled sphingomyelin species

We present a method of analyzing sphingomyelin (SM) qualitatively and quantitatively by liquid chromatography-electrospray Ionization-tandem mass spectrometry (LC-ESI-MS/MS). SM is a common sphingolipid composed of a phosphorylcholine and a ceramide as the hydrophilic and hydrophobic component, respectively. A number of SM species are present in mammalian cells due to a variety in the sphingoid long chain base (LCB) and an N-acyl moiety in the ceramide. In this report, we show a method of estimating the number of carbon and double bonds in a LCB and an N-acyl moiety based on their corresponding product ions in MS/MS/MS (MS3) experiments. In addition, we present a quantitative analysis method for SM using two stable isotopically labeled SM species, which facilitates determining the range used in SM quantitation. The present method will be useful in characterizing a variety of SM species in biological samples and industrial products such as cosmetics.

Chemically synthesized d18:1/24:0 SM (Figure 1A) and d18:1/24:0 SM in lipid samples extracted from HeLa cells (Figure 1B) were analyzed by LC-ESI-MS3 employing [M+HCOO]- and [M-CH3]- as first and second precursor ions, respectively. Note that the spectrum intensity of demethylated-sphingosylphosphorylcholine (SPC) (m/z 449) is larger than that ...

Watch this Scientific Journal Video about Quantitative and Qualitative Method for Sphingomyelin by LC-MS Using Two Stable Isotopically Labeled Sphingomyelin Species at JoVE.com

Quantitative and Qualitative Method for Sphingomyelin by LC-MS Using Two Stable Isotopically Labeled Sphingomyelin Species

Here, we present a protocol to quantify and qualify each sphingomyelin species using multiple reaction monitoring and MS/MS/MS mode, respectively.

We present a method of analyzing sphingomyelin (SM) qualitatively and quantitatively by liquid chromatography-electrospray Ionization-tandem mass ...

quantitative-qualitative-method-for-sphingomyelin-lc-ms-using-two

Universidad de Cartagena UDEC

Research

JoVE Journal

Biochemistry

9.7K Views.  Teikyo University. Here, we present a protocol to quantify and qualify each sphingomyelin species using multiple reaction monitoring and MS/MS/MS mode, respectively.

Video: Quantitative and Qualitative Method for Sphingomyelin by LC-MS Using Two Stable Isotopically Labeled Sphingomyelin Species

An Efficient Method for the Isolation of Highly Purified RNA from Seeds for Use in Quantitative Transcriptome Analysis

Plant seeds accumulate large amounts of storage reserves comprising biodegradable organic matter. Humans rely on seed storage reserves for food and as industrial materials. Gene expression profiles are powerful tools for investigating metabolic regulation in plant cells. Therefore, detailed, accurate gene expression profiles during seed development are required for crop breeding. Acquiring highly purified RNA is essential for producing these profiles. Efficient methods are needed to isolate highly purified RNA from seeds. Here, we describe a method for isolating RNA from seeds containing large amounts of oils, proteins, and polyphenols, which have inhibitory effects on high-purity RNA isolation. Our method enables highly purified RNA to be obtained from seeds without the use of phenol, chloroform, or additional processes for RNA purification. This method is applicable to Arabidopsis, rapeseed, and soybean seeds. Our method will be useful for monitoring the expression patterns of low level transcripts in developing and mature seeds.

We have succeeded in establishing a method for RNA isolation from plant seeds containing large amounts of oils, proteins, and polyphenols, which have inhibitory effects on high-purity RNA isolation. Our method is suitable for monitoring the expression of genes with low level transcripts in seeds.

Plant seeds accumulate large amounts of storage reserves comprising biodegradable organic matter. Humans rely on seed storage reserves for food and as ...

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 Protein Preparation Method for the High-throughput Identification of Proteins Interacting with a Nuclear Cofactor Using LC-MS/MS Analysis

Transcriptional coregulators are vital to the efficient transcriptional regulation of nuclear chromatin structure. Coregulators play a variety of roles in regulating transcription. These include the direct interaction with transcription factors, the covalent modification of histones and other proteins, and the occasional chromatin conformation alteration. Accordingly, establishing relatively quick methods for identifying proteins that interact within this network is crucial to enhancing our understanding of the underlying regulatory mechanisms. LC-MS/MS-mediated protein binding partner identification is a validated technique used to analyze protein-protein interactions. By immunoprecipitating a previously-identified member of a protein complex with an antibody (occasionally with an antibody for a tagged protein), it is possible to identify its unknown protein interactions via mass spectrometry analysis. Here, we present a method of protein preparation for the LC-MS/MS-mediated high-throughput identification of protein interactions involving nuclear cofactors and their binding partners. This method allows for a better understanding of the transcriptional regulatory mechanisms of the targeted nuclear factors.

We have established a method for the purification of coregulatory interaction proteins using the LC-MS/MS system.

Transcriptional coregulators are vital to the efficient transcriptional regulation of nuclear chromatin structure. Coregulators play a variety of roles in ...

DNA Polymerase Activity Assay Using Near-infrared Fluorescent Labeled DNA Visualized by Acrylamide Gel Electrophoresis

For any enzyme, robust, quantitative methods are required for characterization of both native and engineered enzymes. For DNA polymerases, DNA synthesis can be characterized using an in vitro DNA synthesis assay followed by polyacrylamide gel electrophoresis. The goal of this assay is to quantify synthesis of both natural DNA and modified DNA (M-DNA). These approaches are particularly useful for resolving oligonucleotides with single nucleotide resolution, enabling observation of individual steps during enzymatic oligonucleotide synthesis. These methods have been applied to the evaluation of an array of biochemical and biophysical properties such as the measurement of steady-state rate constants of individual steps of DNA synthesis, the error rate of DNA synthesis, and DNA binding affinity. By using modified components including, but not limited to, modified nucleoside triphosphates (NTP), M-DNA, and/or mutant DNA polymerases, the relative utility of substrate-DNA polymerase pairs can be effectively evaluated. Here, we detail the assay itself, including the changes that must be made to accommodate nontraditional primer DNA labeling strategies such as near-infrared fluorescently labeled DNA. Additionally, we have detailed crucial technical steps for acrylamide gel pouring and running, which can often be technically challenging.

This protocol describes the characterization of DNA polymerase synthesis of modified DNA through observation of changes to near-infrared fluorescently labeled DNA using gel electrophoresis and gel imaging. Acrylamide gels are used for high resolution imaging of the separation of short nucleic acids, which migrate at different rates depending on size.

For any enzyme, robust, quantitative methods are required for characterization of both native and engineered enzymes. For DNA polymerases, DNA synthesis ...

Highly Sensitive and Quantitative Detection of Proteins and Their Isoforms by Capillary Isoelectric Focusing Method

Immunoblotting has become a routine technique in many laboratories for protein characterization from biological samples. The following protocol provides an alternative strategy, capillary isoelectric focusing (cIEF), with many advantages compared to conventional immunoblotting. This is an antibody-based, automated, rapid, and quantitative method in which a complete western blotting procedure takes place inside an ultrathin capillary. This technique does not require a gel to transfer to a membrane, stripping of blots, or x-ray films, which are typically required for conventional immunoblotting. Here, proteins are separated according to their charge (isoelectric point; pI), using less than a microliter (400 nL) of total protein lysate. After electrophoresis, proteins are immobilized onto the capillary walls by ultraviolet light treatment, followed by primary and secondary (horseradish peroxidase (HRP) conjugated) antibody incubation, whose binding is detected through enhanced chemiluminescence (ECL), generating a light signal that can be captured and recorded by a charge-coupled device (CCD) camera. The digital image can be analyzed and quantified (peak area) using software. This high throughput procedure can handle 96 samples at a time; is highly sensitive, with protein detection in the picogram range; and produces highly reproducible results because of automation. All of these aspects are extremely valuable when the quantity of samples (e.g., tissue samples and biopsies) is a limiting factor. The technique has wider applications as well, including screening of drugs or antibodies, biomarker discovery, and diagnostic purposes.

Capillary isoelectric focusing is an antibody-based, ultrasensitive, high throughput technique, enabling detailed characterization of proteins and their isoforms from extremely small biological samples. The following describes a protocol for detection and quantification of specific proteins and their isoforms in an automated and robotized manner.

Immunoblotting has become a routine technique in many laboratories for protein characterization from biological samples. The following protocol provides ...

Fractionation for Resolution of Soluble and Insoluble Huntingtin Species

The accumulation of misfolded proteins is central to pathology in Huntington's disease (HD) and many other neurodegenerative disorders. Specifically, a key pathological feature of HD is the aberrant accumulation of mutant HTT (mHTT) protein into high molecular weight complexes and intracellular inclusion bodies composed of fragments and other proteins. Conventional methods to measure and understand the contributions of various forms of mHTT-containing aggregates include fluorescence microscopy, western blot analysis, and filter trap assays. 
However, most of these methods are conformation specific, and therefore may not resolve the full state of mHTT protein flux due to the complex nature of aggregate solubility and resolution.
For the identification of aggregated mHTT and various modified forms and complexes, separation and solubilization of the cellular aggregates and fragments is mandatory. Here we describe a method to isolate and visualize soluble mHTT, monomers, oligomers, fragments, and an insoluble high molecular weight (HMW) accumulated mHTT species. HMW mHTT tracks with disease progression, corresponds with mouse behavior readouts, and has been beneficially modulated by certain therapeutic interventions1. This approach can be used with mouse brain, peripheral tissues, and cell culture but may be adapted to other model systems or disease contexts.

A method is described for fractionation of insoluble and soluble mutant huntingtin species from mouse brain and cell culture. The method described is useful for characterization and quantification of huntingtin protein flux and aids in analyzing protein homeostasis in disease pathogenesis and in the presence of perturbations

The accumulation of misfolded proteins is central to pathology in Huntington's disease (HD) and many other neurodegenerative disorders. Specifically, a ...

Efficient Purification and LC-MS/MS-based Assay Development for Ten-Eleven Translocation-2 5-Methylcytosine Dioxygenase

The epigenetic transcription regulation mediated by 5-methylcytosine (5mC) has played a critical role in eukaryotic development. Demethylation of these epigenetic marks is accomplished by sequential oxidation by ten-eleven translocation dioxygenases (TET1-3), followed by the thymine-DNA glycosylase-dependent base excision repair. Inactivation of the TET2 gene due to genetic mutations or by other epigenetic mechanisms is associated with a poor prognosis in patients with diverse cancers, especially hematopoietic malignancies. Here, we describe an efficient single step purification of enzymatically active untagged human TET2 dioxygenase using cation exchange chromatography. We further provide a liquid chromatography-tandem mass spectrometry (LC-MS/MS) approach that can separate and quantify the four normal DNA bases (A, T, G, and C), as well as the four modified cytosine bases (5-methyl, 5-hydroxymethyl, 5-formyl, and 5-carboxyl). This assay can be used to evaluate the activity of wild type and mutant TET2 dioxygenases.

Here, we present a protocol for an efficient single step purification of the active untagged human ten-eleven translocation-2 (TET2) 5-methylcytosine dioxygenase using ion-exchange chromatography and its assay using a liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based approach.

The epigenetic transcription regulation mediated by 5-methylcytosine (5mC) has played a critical role in eukaryotic development. Demethylation of these ...

Robust Comparison of Protein Levels Across Tissues and Throughout Development Using Standardized Quantitative Western Blotting

Western blotting is a technique that is commonly used to detect and quantify protein expression. Over the years, this technique has led to many advances in both basic and clinical research. However, as with many similar experimental techniques, the outcome of Western blot analyses is easily influenced by choices made in the design and execution of the experiment. Specific housekeeping proteins have traditionally been used to normalize protein levels for quantification, however, these have a number of limitations and have therefore been increasingly criticized over the past few years. Here, we describe a detailed protocol that we have developed to allow us to undertake complex comparisons of protein expression variation across different tissues, mouse models (including disease models), and developmental timepoints. By using a fluorescent total protein stain and introducing the use of an internal loading standard, it is possible to overcome existing limitations in the number of samples that can be compared within experiments and systematically compare protein levels across a range of experimental conditions. This approach expands the use of traditional western blot techniques, thereby allowing researchers to better explore protein expression across different tissues and samples.

This method describes a robust and reproducible approach for the comparison of protein levels in different tissues and at different developmental timepoints using a standardized quantitative western blotting approach.

Western blotting is a technique that is commonly used to detect and quantify protein expression. Over the years, this technique has led to many advances ...

Ubiquitin Chain Analysis by Parallel Reaction Monitoring

Assessment of the global profile of ubiquitin chain topologies within a proteome is of interest to answer a wide range of biological questions. The protocol outlined here takes advantage of the di-glycine (-GG) modification left after the tryptic digestion of ubiquitin incorporated in a chain. By quantifying these topology-characteristic peptides the relative abundance of each ubiquitin chain topology can be determined. The steps required to quantify these peptides by a parallel reaction monitoring experiment are reported taking into consideration the stabilization of ubiquitin chains. Preparation of heavy controls, cell lysis, and digestion are described along with the appropriate mass spectrometer setup and data analysis workflow. An example data set with perturbations in ubiquitin topology is presented, accompanied by examples of how optimization of the protocol can affect results. By following the steps outlined, a user will be able to perform a global assessment of the ubiquitin topology landscape within their biological context.

This method describes the assessment of global changes in ubiquitin chain topology. The assessment is performed by the application of a mass spectrometry-based targeted proteomics approach.

Assessment of the global profile of ubiquitin chain topologies within a proteome is of interest to answer a wide range of biological questions. The ...

Single-Molecule Imaging of EWS-FLI1 Condensates Assembling on DNA

The fusion genes resulting from chromosomal translocation have been found in many solid tumors or leukemia. EWS-FLI1, which belongs to the FUS/EWS/TAF15 (FET) family of fusion oncoproteins, is one of the most frequently involved fusion genes in Ewing sarcoma. These FET family fusion proteins typically harbor a low-complexity domain (LCD) of FET protein at their N-terminus and a DNA-binding domain (DBD) at their C-terminus. EWS-FLI1 has been confirmed to form biomolecular condensates at its target binding loci due to LCD-LCD and LCD-DBD interactions, and these condensates can recruit RNA polymerase II to enhance gene transcription. However, how these condensates are assembled at their binding sites remains unclear. Recently, a single-molecule biophysics method-DNA Curtains-was applied to visualize these assembling processes of EWS-FLI1 condensates. Here, the detailed experimental protocol and data analysis approaches are discussed for the application of DNA Curtains in studying the biomolecular condensates assembling on target DNA.

Here, we describe the use of the single-molecule imaging method, DNA Curtains, to study the biophysical mechanism of EWS-FLI1 condensates assembling on DNA.

The fusion genes resulting from chromosomal translocation have been found in many solid tumors or leukemia. EWS-FLI1, which belongs to the FUS/EWS/TAF15 ...