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

Dette arbeidet ble støttet av et forskningsstipend fra Kunnskapsdepartementet, kultur, sport, vitenskap og teknologi i Japan (KAKENHI) å grunnlegge Vennskapelighetsstiftelsen (#15 K 01691), Y.F. (#15 K 08625), K.Y. (#26461532) og stipend for studier av vanskelige sykdom prosjektet fra departementet for Helse, Arbeiderpartiet og velferd (K.Y. #201510032A). Vi takker gruppen Edanz (www.edanzediting.com/ac) for å redigere et utkast av dette manuskriptet.

Se alle relevante sikkerhetsdatablader (MSDS) før bruk. Bruke hansker for å minimere eksempel forurensning av hud-avledet SM. Nåværende protokollen ble brukt HeLa celler dyrket i Eagle's minimum viktig medium supplert med 10% fosterets bovin serum (FBS), 2 mM L-glutamin, 1000 U/L penicillin og 100 mg/L streptomycin.
1. forberedelse av Lipid prøver
Merk: Det er viktig at alle glass inkludert reagensglass med Teflon-lined skrulokk være vaskemiddel uten.</...

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	<li>Huitema, K., van den Dikkenberg, J., Brouwers, J. F., Holthuis, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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. H., Sullards, M. C., Allegood, J. C., Kelly, S., Wang, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sphingolipidomics:+high-throughput,+structure-specific,+and+quantitative+analysis+of+sphingolipids+by+liquid+chromatography+tandem+mass+spectrometry.">Sphingolipidomics: high-throughput, structure-specific, and quantitative analysis of sphingolipids by liquid chromatography tandem mass spectrometry.</a> Methods. 36 (2), 207-224 (2005).</li><li>Houjou, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+and+selective+identification+of+molecular+species+in+phosphatidylcholine+and+sphingomyelin+by+conditional+neutral+loss+scanning+and+MS3.">Rapid and selective identification of molecular species in phosphatidylcholine and sphingomyelin by conditional neutral loss scanning and MS3.</a> Rapid Commun Mass Spectrom. 18 (24), 3123-3130 (2004).</li><li>Hama, K., Fujiwara, Y., Tabata, H., Takahashi, H., Yokoyama, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+Quantitation+Using+Two+Stable+Isotopically+Labeled+Species+and+Direct+Detection+of+N-Acyl+Moiety+of+Sphingomyelin.">Comprehensive Quantitation Using Two Stable Isotopically Labeled Species and Direct Detection of N-Acyl Moiety of Sphingomyelin.</a> Lipids. , (2017).</li><li>Bligh, E. G., Dyer, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+method+of+total+lipid+extraction+and+purification.">A rapid method of total lipid extraction and purification.</a> Can. J. Biochem. Physiol. 37, 911-917 (1959).</li><li>Gu, H., Liu, G., Wang, J., Aubry, A. F., Arnold, M. 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I den nåværende kvalitative metoden, vi fikk MS3 produkt ioner en SPC og en N-acyl moiety. Det er avgjørende for riktig tilordne både et SPC og en N-acyl moiety. For dette formål, bør det bemerkes at andre phosphorylcholine inneholder molekyler også kan gjenkjennes som MS3 produkt ioner. Diacyl-phosphatidylcholine (PC) og plasmalogen-PC finnes rikelig i pattedyrceller, og deres hydrophobicity er lik som SM. Derfor kan diacyl-PC og plasmalogen-PC med en isotop (vanligvis <sup...

Sphingomyelin (SM) er en felles sphingolipid i pattedyrceller. SM er syntetisert intracellulært1 og gave som en forløper til andre sphingolipider som en sphingosine-1-fosfat og en ceramide, som har avgjørende roller i immun celle smugling og huden barriere homeostase, henholdsvis2, 3. Dermed er nøyaktig analyse av SM metabolismen viktig for Klargjørende fysiologiske og patologiske rollene til sphingolipider.
SM består av en ceramide og en phosphorylcholine som er knyttet til gruppen 1-hydroxy ceramide, som ytterligere består av en sphingosine og en N-acyl moiety. En variasjon i karbon og dobbel bond tallene i både sphingosine og N-acyl moiety resulterer i antall ceramide (og SM) arter. Nylige fremskritt innen LC-ESI-MS/MULTIPLE Sclerosis har aktivert kvantitative og kvalitative analyser av SM4,5. I kvalitativ analyse, ble antall karbon og dobbelt bånd en sphingoid LCB av SM identifisert ved å tilordne produktet ion spektra av LCB. Imidlertid strukturinformasjon av N-acyl moiety var ikke direkte fått fordi dens tilsvarende fabrikat ioner ikke er rapportert, og derfor N-acyl moieties ble utledet av differensialanalyse mellom forløper ioner og produktet ioner tilsvarer LCB i både positive og negative ion moduser4,5. I denne rapporten presenterer vi en metode for å oppdage de produkt ionene LCB og N-acyl moiety samtidig i MS3 modus med tre quadrupole og quadrupole lineær ion felle massespektrometri, som muliggjør nøyaktig strukturelle spekulasjon hver SM arter6.
Ion undertrykkelse (eller forbedring) effekten forårsaket av matrix i biologiske prøver hemme nøyaktig kvantifiseringen LC-ESI-MULTIPLE Sclerosis analyse, og derfor er det ønskelig å konstruere kalibrering kurver for alle analytter rundt i identiske matrix av biologiske utvalget. Men er denne strategien ikke mulig fordi det er nesten umulig å forberede alle SM arter i biologiske prøver, spesielt i omfattende analyse. Dermed er det praktisk å konstruere en kalibreringskurven og bestemme kvantitative området som bruker en representant SM Art piggete i de biologiske prøvene. Vi brukte to isotopically-merket SM arter for å konstruere en kalibreringskurven; en ble brukt til en intern standard og den andre for en standard sammensatte. Vi oppdaget litt isotopically-merket SM arter som en standard compound piggete i biologiske prøver og vellykket innhentet en kalibreringskurven og kvantitative utvalg6.

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		<col />
		<col />
	</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

Vi presenterer en metode til å analysere sphingomyelin (SM) kvalitativt og kvantitativt ved flytende kromatografi-electrospray Ionization-tandem massespektrometri (LC-ESI-MS/MS). SM er en vanlig sphingolipid består av en phosphorylcholine og en ceramide som de hydrofile og hydrofobe komponent henholdsvis. En rekke SM arter finnes i pattedyrceller av i sphingoid lange kjeden base (LCB) og en N-acyl moiety i ceramide. I denne rapporten, viser vi en metode for å beregne antall karbon og dobbel obligasjoner i en LCB og en N-acyl moiety basert på deres tilsvarende produkt ioner i MS/MS/MS (MS3) eksperimenter. I tillegg presenterer vi en kvantitativ analysemetode for SM bruker to stabile isotopically merket SM arter som forenkler bestemme området brukes i SM kvantifisering. Metoden stede vil være nyttig i karakterisere en rekke SM arter i biologiske prøver og industrielle produkter som kosmetikk.

Syntetisk kjemisk d18:1 / 24:0 SM (figur 1A) og d18:1 / 24:0 SM i lipid eksempler Hentet fra HeLa celler (figur 1B) ble analysert av LC-ESI-MS3 ansette [M + HCOO]- og [M-CH3]- som første og andre forløper ioner, henholdsvis. Spekteret intensiteten av demethylated-sphingosylphosphorylcholine (SPC) (m/z 449) er større enn SM N-acyl mo...

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

Kvantitative og kvalitative metode for Sphingomyelin av LC-MS bruker to stabil Isotopically merket Sphingomyelin arter

Her presenterer vi en protokoll for å kvantifisere og kvalifisere hver sphingomyelin Art bruker flere reaksjon overvåking og MS-/ MS-/ MS modus, henholdsvis.

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

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Enkeltmolekylavbildning av EWS-FLI1-kondensater som monteres på 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

En robust kryo-elektronmikroskopi (cryo-EM) prosesseringsarbeidsflyt med kryoSPARC, RELION og 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

Analysere samspillet mellom fluorescerende merkede proteiner med kunstige fosfolipidmikrovesikler ved hjelp av kvantitativ strømningscytometri

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 ...