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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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.

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

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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

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 Optimized Protocol for Electrophoretic Mobility Shift Assay Using Infrared Fluorescent Dye-labeled Oligonucleotides

Electrophoretic Mobility Shift Assays (EMSA) are an instrumental tool to characterize the interactions between proteins and their target DNA sequences. Radioactivity has been the predominant method of DNA labeling in EMSAs. However, recent advances in fluorescent dyes and scanning methods have prompted the use of fluorescent tagging of DNA as an alternative to radioactivity for the advantages of easy handling, saving time, reducing cost, and improving safety. We have recently used fluorescent EMSA (fEMSA) to successfully address an important biological question. Our fEMSA analysis provides mechanistic insight into the effect of a missense mutation, G73E, in the highly conserved HMG transcription factor SOX-2 on olfactory neuron type diversification. We found that mutant SOX-2G73E protein alters specific DNA binding activity, thereby causing olfactory neuron identity transformation. Here, we present an optimized and cost-effective step-by-step protocol for fEMSA using infrared fluorescent dye-labeled oligonucleotides containing the LIM-4/SOX-2 adjacent target sites and purified SOX-2 proteins (WT and mutant SOX-2G73E proteins) as a biological example.

We describe here an optimized protocol of fluorescent Electrophoretic Mobility Shift Assays (fEMSA) using purified SOX-2 proteins together with infrared fluorescent dye-labeled DNA probes as a case study to tackle an important biological question.

Electrophoretic Mobility Shift Assays (EMSA) are an instrumental tool to characterize the interactions between proteins and their target DNA sequences. ...

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. ...

Cellular Lipid Extraction for Targeted Stable Isotope Dilution Liquid Chromatography-Mass Spectrometry Analysis

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including numerous stereoisomers1,2. These metabolites can be formed from free or esterified fatty acids. Many of these oxidized metabolites have biological activity and have been implicated in various diseases including cardiovascular and neurodegenerative diseases, asthma, and cancer3-7. Oxidized bioactive lipids can be formed enzymatically or by reactive oxygen species (ROS). Enzymes that metabolize fatty acids include cyclooxygenase (COX), lipoxygenase (LO), and cytochromes P450 (CYPs)1,8. Enzymatic metabolism results in enantioselective formation whereas ROS oxidation results in the racemic formation of products.
While this protocol focuses primarily on the analysis of AA- and some LA-derived bioactive metabolites; it could be easily applied to metabolites of other fatty acids. Bioactive lipids are extracted from cell lysate or media using liquid-liquid (l-l) extraction. At the beginning of the l-l extraction process, stable isotope internal standards are added to account for errors during sample preparation. Stable isotope dilution (SID) also accounts for any differences, such as ion suppression, that metabolites may experience during the mass spectrometry (MS) analysis9. After the extraction, derivatization with an electron capture (EC) reagent, pentafluorylbenzyl bromide (PFB) is employed to increase detection sensitivity10,11. Multiple reaction monitoring (MRM) is used to increase the selectivity of the MS analysis. Before MS analysis, lipids are separated using chiral normal phase high performance liquid chromatography (HPLC). The HPLC conditions are optimized to separate the enantiomers and various stereoisomers of the monitored lipids12. This specific LC-MS method monitors prostaglandins (PGs), isoprostanes (isoPs), hydroxyeicosatetraenoic acids (HETEs), hydroxyoctadecadienoic acids (HODEs), oxoeicosatetraenoic acids (oxoETEs) and oxooctadecadienoic acids (oxoODEs); however, the HPLC and MS parameters can be optimized to include any fatty acid metabolites13.
Most of the currently available bioanalytical methods do not take into account the separate quantification of enantiomers. This is extremely important when trying to deduce whether or not the metabolites were formed enzymatically or by ROS. Additionally, the ratios of the enantiomers may provide evidence for a specific enzymatic pathway of formation. The use of SID allows for accurate quantification of metabolites and accounts for any sample loss during preparation as well as the differences experienced during ionization. Using the PFB electron capture reagent increases the sensitivity of detection by two orders of magnitude over conventional APCI methods. Overall, this method, SID-LC-EC-atmospheric pressure chemical ionization APCI-MRM/MS, is one of the most sensitive, selective, and accurate methods of quantification for bioactive lipids.

This protocol will demonstrate the extraction and analysis of free and esterified bioactive fatty acids from cells. Fatty acids are accurately quantified using stable isotope dilution, chiral liquid chromatography, electron capture atmospheric chemical ionization multiple reaction monitoring mass spectrometry (SID-LC-ECAPCI-MRM/MS).

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including ...

Bioengineering

Lipid Droplet Isolation for Quantitative Mass Spectrometry Analysis

Lipid droplets are vital to the replication of a variety of different pathogens, most prominently the Hepatitis C Virus (HCV), as the putative site of virion morphogenesis. Quantitative lipid droplet proteome analysis can be used to identify proteins that localize to or are displaced from lipid droplets under conditions such as virus infections. Here, we describe a protocol that has been successfully used to characterize the changes in the lipid droplet proteome following infection with HCV. We use Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) and thus label the complete proteome of one population of cells with &#34;heavy&#34; amino acids to quantitate the proteins by mass spectrometry. For lipid droplet isolation, the two cell populations (i.e.&#160;HCV-infected/&#34;light&#34; amino acids and uninfected control/&#34;heavy&#34; amino acids) are mixed 1:1 and lysed mechanically in hypotonic buffer. After removing the nuclei and cell debris by low speed centrifugation, lipid droplet-associated proteins are enriched by two subsequent ultracentrifugation steps followed by three washing steps in isotonic buffer. The purity of the lipid droplet fractions is analyzed by western blotting with antibodies recognizing different subcellular compartments. Lipid droplet-associated proteins are then separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by Coomassie staining. After tryptic digest, the peptides are quantified by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). Using this method, we identified proteins recruited to lipid droplets upon HCV infection that might represent pro- or antiviral host factors. Our method can be applied to a variety of different cells and culture conditions, such as infection with pathogens, environmental stress, or drug treatment.

Lipid droplets are important organelles for the replication of several pathogens, including the Hepatitis C Virus (HCV). We describe a method to isolate lipid droplets for quantitative mass spectrometry of associated proteins; it can be used under a variety of conditions, such as virus infection, environmental stress, or drug treatment.

Lipid droplets are vital to the replication of a variety of different pathogens, most prominently the Hepatitis C Virus (HCV), as the putative site of ...

LC-MS Analysis of Human Platelets as a Platform for Studying Mitochondrial Metabolism

Perturbed mitochondrial metabolism has received renewed interest as playing a causative role in a range of diseases. Probing alterations to metabolic pathways requires a model in which external factors can be well controlled, allowing for reproducible and meaningful results. Many studies employ transformed cellular models for these purposes; however, metabolic reprogramming that occurs in many cancer cell lines may introduce confounding variables. For this reason primary cells are desirable, though attaining adequate biomass for metabolic studies can be challenging. Here we show that human platelets can be utilized as a platform to carry out metabolic studies in combination with liquid chromatography-tandem mass spectrometry analysis. This approach is amenable to relative quantification and isotopic labeling to probe the activity of specific metabolic pathways. Availability of platelets from individual donors or from blood banks makes this model system applicable to clinical studies and feasible to scale up. Here we utilize isolated platelets to confirm previously identified compensatory metabolic shifts in response to the complex I inhibitor rotenone. More specifically, a decrease in glycolysis is accompanied by an increase in fatty acid oxidation to maintain acetyl-CoA levels. Our results show that platelets can be used as an easily accessible and medically relevant model to probe the effects of xenobiotics on cellular metabolism.

Here we show isolated human platelets can be used as an accessible ex vivo model to study metabolic adaptations in response to the complex I inhibitor rotenone. This approach employs isotopic tracing and relative quantification by liquid chromatography-mass spectrometry and can be applied to a variety of study designs.

Perturbed mitochondrial metabolism has received renewed interest as playing a causative role in a range of diseases. Probing alterations to metabolic ...

Environment

Mass Spectrometric Analysis of Glycosphingolipid Antigens

Glycosphingolipids (GSL's) belong to the glycoconjugate class of biomacromolecules, which bear structural information for significant biological processes such as embryonic development, signal transduction, and immune receptor recognition1-2. They contain complex sugar moieties in the form of isomers, and lipid moieties with variations including fatty acyl chain length, unsaturation, and hydroxylation. Both carbohydrate and ceramide portions may be basis of biological significance. For example, tri-hexosylceramides include globotriaosylceramide (Gal&alpha;4Gal&beta;4Glc&beta;1Cer) and isoglobotriaosylceramide (Gal&alpha;3Gal&beta;4Glc&beta;1Cer), which have identical molecular masses but distinct sugar linkages of carbohydrate moiety, responsible for completely different biological functions3-4. In another example, it has been demonstrated that modification of the ceramide part of alpha-galactosylceramide, a potent agonist ligand for invariant NKT cells, changes their cytokine secretion profiles and function in animal models of cancer and auto-immune diseases5. The difficulty in performing a structural analysis of isomers in immune organs and cells serve as a barrier for determining many biological functions6.
Here, we present a visualized version of a method for relatively simple, rapid, and sensitive analysis of glycosphingolipid profiles in immune cells7-9. This method is based on extraction and chemical modification (permethylation, see below Figure 5A, all OH groups of hexose were replaced by MeO after permethylation reaction) of glycosphingolipids10-15, followed by subsequent analysis using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) and ion trap mass spectrometry. This method requires 50 million immune cells for a complete analysis. The experiments can be completed within a week. The relative abundance of the various glycosphingolipids can be delineated by comparison to synthetic standards. This method has a sensitivity of measuring 1% iGb3 among Gb3 isomers, when 2 fmol of total iGb3/Gb3 mixture is present9.
Ion trap mass spectrometry can be used to analyze isomers. For example, to analyze the presence of globotriaosylceramide and isoglobtriaosylceramide in the same sample, one can use the fragmentation of glycosphingolipid molecules to structurally discriminate between the two (see below Figure 5). Furthermore, chemical modification of the sugar moieties (through a permethylation reaction) improves the ionization and fragmentation efficiencies for higher sensitivity and specificity, and increases the stability of sialic acid residues. The extraction and chemical modification of glycosphingolipids can be performed in a classic certified chemical hood, and the mass spectrometry can be performed by core facilities with ion trap MS instruments.

A specific and sensitive method to gain insight into the expression profile of glycosphingolipid antigens in immune organs and cells is described. The method takes advantage of the ion trap mass spectrometry allowing step-wise fragmentation of glycosphingolipid molecules for structural analysis in comparison to chemically synthesized standards.

Glycosphingolipids (GSL's) belong to the glycoconjugate class of biomacromolecules, which bear structural information for significant biological processes ...

Immunology and Infection

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

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

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

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

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

Preparation of Human Tissues Embedded in Optimal Cutting Temperature Compound for Mass Spectrometry Analysis

Sphingolipids are cellular components that have well-established roles in human metabolism and disease. Mass spectrometry can be used to determine whether sphingolipids are altered in a disease and investigate whether sphingolipids can be targeted clinically. However, properly powered prospective studies that acquire tissues directly from the surgical suite can be time consuming, and technically, logistically, and administratively challenging. In contrast, retrospective studies can take advantage of cryopreserved human specimens already available, usually in large numbers, at tissue biorepositories. Other advantages of procuring tissues from biorepositories include access to information associated with the tissue specimens including histology, pathology, and in some instances clinicopathological variables, all of which can be used to examine correlations with lipidomics data. However, technical limitations related to the incompatibility of optimal cutting temperature compound (OCT) used in the cryopreservation and mass spectrometry is a technical barrier for the analysis of lipids. However, we have previously shown that OCT can be easily removed from human biorepository specimens through cycles of washes and centrifugation without altering their sphingolipid content. We have also previously established that sphingolipids in human tissues cryopreserved in OCT are stable for up to 16 years. In this report, we outline the steps and workflow to analyze sphingolipids in human tissue specimens that are embedded in OCT, including washing tissues, weighing tissues for data normalization, the extraction of lipids, preparation of samples for analysis by liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS), mass spectrometry data integration, data normalization, and data analysis.

Sphingolipids are bioactive metabolites with well-established roles in human disease. Characterizing alterations in tissues with mass-spectrometry can reveal roles in disease etiology or identify therapeutic targets. However, the OCT-compound used for cryopreservation in biorepositories interferes with mass-spectrometry. We outline methods to analyze sphingolipids in human tissues embedded in OCT with LC-ESI-MS/MS.

Sphingolipids are cellular components that have well-established roles in human metabolism and disease. Mass spectrometry can be used to determine whether ...

Cancer Research

Screening Lipid Structures Using Stable Isotope Labeling

Tracing de novo Lipids using Stable Isotope Labeling LC-TIMS-TOF MS/MS

Lipids are highly diverse, and small changes in lipid structures and composition can have profound effects on critical biological functions. Stable isotope labeling (SIL) offers several advantages for the study of lipid distribution, mobilization, and metabolism, as well as de novo lipid synthesis. The successful implementation of the SIL technique requires the removal of interferences from endogenous molecules. In the present work, we describe a high-throughput analytical protocol for the screening of SIL lipids from biological samples; examples will be shown of lipid de novo identification during mosquito ovary development. The use of complementary liquid chromatography trapped ion mobility spectrometry and mass spectrometry allows for the separation and lipids assignment from a single sample in a single scan (&lt;1 h). The described approach takes advantage of recent developments in data-dependent acquisition and data-independent acquisition, using parallel accumulation in the mobility trap followed by sequential fragmentation and collision-induced dissociation. The measurement of SIL at the fatty acid chain level reveals changes in lipid dynamics during the ovary development of mosquitoes. The lipids de novo structures are confidently assigned based on their retention time, mobility, and fragmentation pattern.

Author Spotlight: Quantification of Complex Lipidomic Samples Using Stable Isotope Labeling

Presented here is a method for screening lipid structures through stable isotope labeling by using their retention time, mobility, and fragmentation.

Lipids are highly diverse, and small changes in lipid structures and composition can have profound effects on critical biological functions. Stable ...

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

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An Optimized Protocol for Electrophoretic Mobility Shift Assay Using Infrared Fluorescent Dye-labeled Oligonucleotides

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

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

Cellular Lipid Extraction for Targeted Stable Isotope Dilution Liquid Chromatography-Mass Spectrometry Analysis

Lipid Droplet Isolation for Quantitative Mass Spectrometry Analysis

LC-MS Analysis of Human Platelets as a Platform for Studying Mitochondrial Metabolism

Mass Spectrometric Analysis of Glycosphingolipid Antigens

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

Preparation of Human Tissues Embedded in Optimal Cutting Temperature Compound for Mass Spectrometry Analysis

Tracing de novo Lipids using Stable Isotope Labeling LC-TIMS-TOF MS/MS