Name: quantitative and qualitative method for sphingomyelin by lc-ms using two stable isotopically labeled sphingomyelin species
Uploaded: 2018-05-07T13:00:00
Description: 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

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...

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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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	</colgroup>
	<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 ...

The overall goal of this procedure is to precisely analyze the amount and the structure of each Sphingomyelin species in biological samples. This method can help answer key questions in the lipid field pertaining to lipid biology and lipid The main advantage of this technique is that we can characterize a variety of Sphingomyelin species in biological samples by chromatography mass spectrometry system. To begin, remove the culture media from a 10 centimeter tissue culture dish containing HeLa cells and rinse the cells twice with six milliliters of ice cold PBS.Then add one milliliter of the ice cold PBS into the 10 centimeter tissue culture dish and use a cell scraper to harvest the cells. Once removed from the dish's surface, transfer the cells into two milliliter siliconized plastic tubes. Following centrifugation, remove the supernatant and add one milliliter methanol to each cell pellet.Then briefly vortex the tubes. Next, place the samples into a bath-type sonicator and sonicate at 200 watts for five minutes. When finished, transfer the whole cell homogenate into test tubes equipped with Teflon lined screw caps.Now add one milliliter of methanol, one milliliter of chloroform, 0.8 milliliters of double distilled water in 50 microliters of 10 micromolar of an internal standard into each test tube. Cap the tubes and vortex them vigorously at 2, 500 rpm for five minutes at room temperature. Then add an additional one milliliter of chloroform and one milliliter of double distilled water to each tube.Again, vortex the tubes vigorously at 2, 500 rpm for five minutes at room temperature. Next, centrifuge the samples in order to completely separate the aqueous and organic phases. Transfer the lower phase into disposable glass tubes.Add two milliliters of chloroform into the test tubes. Then vortex the tubes vigorously at 2, 500 rpm for five minutes at room temperature to sufficiently mix with the upper phase and interphasial fluff. Following a second centrifugation, transfer the modified lower phase into the disposable glass tubes along with the initial lower phase.Now place the glass tubes under a nitrogen stream for approximately 60 minutes and completely remove the organic solvents in the collected lower phase. Finally, reconstitute the samples with 500 microliters of methanol or ethanol. Filter the resulting mixture with a 0.02 micron filter into glass vials.Then store the sample at minus 20 degrees Celsius. Perform serial dilutions of the standard compound and add 50 microliters of each concentration into separate test tubes. Next, prepare three quality control samples by adding an amount within three times the lower boundary of the standard curve to the first sample, an amount near the center of the curve for the second sample, and an amount near the upper boundary of the standard curve for the third sample.Then add the cell homogenate in one milliliter of methanol into each test tube and extract the total lipid fraction from each sample using the method just shown. To begin, prepare the mobile phase. Mix together acetonitrile, methanol, and double distilled water at a two to two to one ratio for the aqueous phase.Use isopropanol for the organic phase in glass bottles with Teflon lined screw caps. Then sonicate both of the mobile phases for five minutes in a bath-type sonicator. Then add formic acid to a final concentration of 26.4 millimolar and ammonium hydroxide at 14.9 millimolar into each mobile phase.For qualitative analysis, activate the high performance liquid chromatography system. Put inlet tubes into the glass bottles that contain the mobile phases and purge the HPLC lines. Then link a C18 HPLC column to the HPLC system.Keep the temperature in the column oven at 50 degrees Celsius and condition the column with the aqueous mobile phase at 100 microliters per minute. During the run, place the samples into a sample rack of the auto sampler. Set the parameters of the triple quadruple and quadruple linear ion trap mass spectrometry system for three-stage mass spectrometry analysis as listed in table one and table two of the accompanying text protocol.Also, set the parameters for the first and second precursor ions of the SM species of interest as described in more detail in the accompanying text protocol. Create a batch file and submit the batch file to obtain the data of MS three product ion spectra of each Sphingomyelin species by liquid chromatography electrospray ionization tandem mass spectrometric analysis. Obtain the three-phase MS product ion spectra of the Sphingomyelin species of interest by liquid chromatography electrospray ionization tandem mass spectrometry analysis.Then assign each MS three product ion spectra of the Sphingomyelin by comparing the mass to charge ratio of the product ion spectra in the exact mass of the Sphingoid long-chain base and n-acyl moiety of interest. Quantitatively analyze the Sphingomyelin using liquid chromatography electrospray ionization tandem mass spectrometry analysis as described in the accompanying text protocol. Then process the multiple reaction monitoring data using the software for data integration to obtain the data of the peak area for the extracted ion chromatogram of each Sphingomyelin species.Quantify each Sphingomyelin species and construct the standard curves as described in the accompanying text protocol. The spectrum of both chemically synthesized Sphingomyelin and Sphingomyelin in lipid samples extracted from HeLa cells are shown here. Of note, the spectrum intensity of demethylated sphingosylphosphorylcholine is larger than that of the SM n-acyl moiety in both samples.Also of interest, the spectrum corresponding to sphingosine-1-phosphate is useful to speculate the number of carbon and double bonds of the sphingoid long-chain base of the Sphingomyelin. In the present quantitative method, it is critical to precisely prepare samples for constructing the calibration curve in validating this method. The curve fitting up low concentration was clearly improved by using the weighting factor equals one over x squared as compared with that using the weighting factor of one or one over x.This technique paved the way for researchers in the field of biology and industry to characterize a variety of Sphingomyelin species in biological samples and industry products such as cosmetics. After watching this video, you should have a good understanding of how to precisely analyze the amount and the structure of each Sphingomyelin species in biological samples. Don't forget that working with absorbents can be extremely hazardous and precautions such as using proper glass wears should always be taken while performing this procedure.In addition, it is important to wear gloves to prevent the contamination of lipids derived from your hands.

Research

JoVE Journal

Biochemistry

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

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

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 ...

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 ...

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 ...

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 ...

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 ...

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 ...

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 ...

A Robust Single-Particle Cryo-Electron Microscopy (cryo-EM) Processing Workflow with cryoSPARC, RELION, and Scipion

A Robust Single-Particle Cryo-Electron Microscopy cryo-EM Processing Workflow with cryoSPARC, RELION, and Scipion

Recent advances in both instrumentation and image processing software have made single-particle cryo-electron microscopy (cryo-EM) the preferred method ...

Analyzing the Interaction of Fluorescent-Labeled Proteins with Artificial Phospholipid Microvesicles using Quantitative Flow Cytometry

In the human body, most of the major physiologic reactions involved in the immune response and blood coagulation proceed on the membranes of cells. An ...