Name: lipid droplet isolation for quantitative mass spectrometry analysis
Uploaded: 2017-04-17T13:00:00
Description: Watch this Scientific Journal Video about Lipid Droplet Isolation for Quantitative Mass Spectrometry Analysis at JoVE.com

Vi takker R. Bartenschlager (University of Heidelberg) for de JC1 konstruktioner, CM Rice (Rockefeller University) for Huh7.5 cellerne, J. McLauchlan (Medical Research Council Virologi Unit) til JFH1 konstruere, T. Wakita (National Institute of Infectious Diseases, Japan) til JFH1, og B. Webster og toilet Greene (Gladstone Institute of Virology og Immunologi) for HCVcc reporterkonstruktioner. Dette arbejde blev støttet af midler fra DFG (HE 6889 / 2-1 (EH), INST 337 / 15-1 2013 og INST 337 / 16-1 2013 (HS)). Den Heinrich Pette Institut, Leibniz Institute for Eksperimentel Virologi er støttet af Freie und Hansestadt Hamburg og Forbundsrepublikken sundhedsministerium. De finansieringskilderne havde ingen rolle i studie design, indsamling og analyse af data, beslutning om at offentliggøre, eller forberedelse af manuskriptet.

1. Fremstilling af Media for Stable Isotope Mærkning med aminosyrer i cellekultur (SILAC) BEMÆRK: Her SILAC Protein Kvantificering Kit - DMEM suppleret med 50 mg 13C 6 L-arginin-HCI blev anvendt til SILAC mærkning. Den dialyserede kalvefosterserum (FCS) er forsynet med SILAC Protein Kvantificering Kit. <ol><li> Fjerne 50 ml fra hver flaske DMEM-medium og tilsættes 50 ml dialyseret FCS. </li><li> Opløs 50 mg af 13C 6 L-lysin-2HCI og 50 mg <su...

<ol>
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The perilipin family of structural lipid droplet proteins: stabilization of lipid droplets and control of lipolysis.</a> J Lipid Res. 48 (12), 2547-2559 (2007).</li><li>Choudhary, V., Ojha, N., Golden, A., Prinz, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+conserved+family+of+proteins+facilitates+nascent+lipid+droplet+budding+from+the+ER.">A conserved family of proteins facilitates nascent lipid droplet budding from the ER.</a> J Cell Biol. 211 (2), 261-271 (2015).</li><li>Kory, N., Farese, R. V., Walther, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+Fat:+Mechanisms+of+Protein+Localization+to+Lipid+Droplets.">Targeting Fat: Mechanisms of Protein Localization to Lipid Droplets.</a> Trends Cell Biol. 26 (7), 535-546 (2016).</li><li>Hashemi, H. F., Goodman, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+life+cycle+of+lipid+droplets.">The life cycle of lipid droplets.</a> Curr Opin Cell Biol. 33, 119-124 (2015).</li><li>Gao, Q., Goodman, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+lipid+droplet-a+well-connected+organelle.">The lipid droplet-a well-connected organelle.</a> Front Cell Dev Biol. 3, 49 (2015).</li><li>Herker, E., Ott, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+role+of+lipid+droplets+in+host/pathogen+interactions.">Emerging role of lipid droplets in host/pathogen interactions.</a> J Biol Chem. 287 (4), 2280-2287 (2012).</li><li>Wedemeyer, H., Dore, G. J., Ward, J. 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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=Hepatitis+C+virus+NS5A+colocalizes+with+the+core+protein+on+lipid+droplets+and+interacts+with+apolipoproteins.">Hepatitis C virus NS5A colocalizes with the core protein on lipid droplets and interacts with apolipoproteins.</a> Virology. 292 (2), 198-210 (2002).</li><li>Herker, E., 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=Efficient+hepatitis+C+virus+particle+formation+requires+diacylglycerol+acyltransferase-1.">Efficient hepatitis C virus particle formation requires diacylglycerol acyltransferase-1.</a> Nat Med. 16 (11), 1295-1298 (2010).</li><li>Camus, G., 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=Diacylglycerol+acyltransferase-1+localizes+hepatitis+C+virus+NS5A+protein+to+lipid+droplets+and+enhances+NS5A+interaction+with+the+viral+capsid+core.">Diacylglycerol acyltransferase-1 localizes hepatitis C virus NS5A protein to lipid droplets and enhances NS5A interaction with the viral capsid core.</a> J Biol Chem. 288 (14), 9915-9923 (2013).</li><li>Miyanari, Y., 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=The+lipid+droplet+is+an+important+organelle+for+hepatitis+C+virus+production.">The lipid droplet is an important organelle for hepatitis C virus production.</a> Nat Cell Biol. 9 (9), 1089-1097 (2007).</li><li>Boulant, S., Targett-Adams, P., McLauchlan, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disrupting+the+association+of+hepatitis+C+virus+core+protein+with+lipid+droplets+correlates+with+a+loss+in+production+of+infectious+virus.">Disrupting the association of hepatitis C virus core protein with lipid droplets correlates with a loss in production of infectious virus.</a> J Gen Virol. 88 (Pt 8), 2204-2213 (2007).</li><li>Vogt, D. A., 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=Lipid+droplet-binding+protein+TIP47+regulates+hepatitis+C+Virus+RNA+replication+through+interaction+with+the+viral+NS5A+protein.">Lipid droplet-binding protein TIP47 regulates hepatitis C Virus RNA replication through interaction with the viral NS5A protein.</a> PLoS Pathog. 9 (4), e1003302 (2013).</li><li>Ploen, D., 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=TIP47+plays+a+crucial+role+in+the+life+cycle+of+hepatitis+C+virus.">TIP47 plays a crucial role in the life cycle of hepatitis C virus.</a> J Hepatol. 58 (6), 1081-1088 (2013).</li><li>Ploen, D., 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=TIP47+is+associated+with+the+hepatitis+C+virus+and+its+interaction+with+Rab9+is+required+for+release+of+viral+particles.">TIP47 is associated with the hepatitis C virus and its interaction with Rab9 is required for release of viral particles.</a> Eur J Cell Biol. 92 (12), 374-382 (2013).</li><li>Rosch, K., 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=Quantitative+Lipid+Droplet+Proteome+Analysis+Identifies+Annexin+A3+as+a+Cofactor+for+HCV+Particle+Production.">Quantitative Lipid Droplet Proteome Analysis Identifies Annexin A3 as a Cofactor for HCV Particle Production.</a> Cell Rep. 16 (12), 3219-3231 (2016).</li><li>Kwiatkowski, M., 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=Ultrafast+extraction+of+proteins+from+tissues+using+desorption+by+impulsive+vibrational+excitation.">Ultrafast extraction of proteins from tissues using desorption by impulsive vibrational excitation.</a> Angew Chem Int Ed Engl. 54 (1), 285-288 (2015).</li><li>Webster, B., Ott, M., Greene, W. 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Her beskriver vi en protokol til isolering lipiddråber til kvantitativ lipid dråbe proteomanalyse at sammenligne berigelse og udtømning af proteiner associeret med lipiddråber under forskellige dyrkningsbetingelser, såsom virale infektioner. Som en alternativ fremgangsmåde kan proteomanalyse udføres med label-free kvantificeringer baseret på totale topintensiteter. Denne fremgangsmåde har intet dynamikområde begrænsning og undgår metaboliske problemer. Fordelen ved SILAC tilgang er, at prøverne samles lipid...

<html> Lipiddråber er yderst dynamiske cytoplasmiske (og nukleare) celleorganeller sammensat af en kerne af neutrale lipider (triglycerider (TG) og cholesterolester (CE)) omsluttet af et monolag af phospholipider med indlejrede proteiner 1. Alle celletyper producerer lipiddråber, men de varierer i størrelse, lipidsammensætning og protein dekoration. Lipiddråber opfylder forskellige funktioner, herunder tjener som energi- og membran precursor-reservoirer eller som proteinaflejringer. Desuden gennem optagelsen af lipider, de beskytter celler fra lipotoxicity, frigivelse lipider som signalmolekyler, og er involveret i nedbrydning protein og endoplasmatiske reticulum (ER) stressresponser 2. Som sådan en masse af proteiner binder til lipiddråber og regulerer deres generation, nedbrydning, handel, og interaktion med andre organeller. Blandt dem er den perilipin familie af bona fide lipid dråbe bindingsproteiner (PLIN1-5)f &quot;&gt; 3. Lipid dråbe biogenese starter sandsynligvis på ER, hvor ER-resident enzymer katalyserer syntesen af neutrale lipider, der akkumuleres inden membrandobbeltlaget, danner en linse af neutrale lipider, en proces, der for nylig blev visualiseret pænt i gær 4. Membran bøjning og forhøjede phosphatidsyre og diacylglycerol niveauer er så menes at tiltrække proteiner involveret i phospholipid biosyntese, som samtidig syntese af de centrale neutrale lipider og afskærmning phospholipider er påkrævet for lipid dråbe generation 5. Enzymer, der huser transmembrane domæner, der findes på skadestuen katalyserer denne proces. Ekspansion til store lipiddråber kræver aktiviteten af ​​en anden klasse af lipid-syntese enzymer, der huser en amfipatisk helix og kan således rejse fra ER til lipiddråber. Mobiliseringen af ​​lipider fra lipiddråber sker gennem den lokale aktivering af triglyceridoliene og diacylglycerol lipaser adipose triglycerid lipase (ATGL) og hormon-sensitive lipase (HSL) eller af forskellige autophagic veje, såsom makro- og microlipophagy eller chaperone-medieret autofagi 6. Lipiddråber interagere med andre cellulære organeller, såsom mitochondrier (for beta-oxidation og lipidsyntese) og ER (for lipidsyntese og protein trafficking), men også med lysosomer, endosomer og vakuolerne induceret af intracellulære bakterier 7. Faktisk bakterier, vira og endda parasitter målrette lipid dråber for replikation og vedholdenhed, blandt dem HCV 8. HCV-infektion er en af de førende årsager til leveren sygelighed og dødelighed på verdensplan, der tegner sig for ca. 0,5 millioner dødsfald om året 9. Den sande antal HCV-infektioner er ukendt, men de seneste skøn tyder på, at 130 - 150 millioner mennesker er kronisk smittet. ingen vaccineeksisterer e, men de nyligt godkendte direkte virkende antivirale dramatisk øge terapeutiske respons sammenlignet med standard interferon-baseret behandling. Men på verdensplan, behandlingen af ​​patienter vil sandsynligvis være begrænset på grund af de ekstremt høje omkostninger ved de nye lægemidler. Omkring halvdelen af ​​alle personer kronisk inficeret med HCV udvikle fedtlever (steatosis), en tilstand kendetegnet ved overdreven ophobning af lipiddråber i hepatocytter. Interessant lipiddråber opstod også som vitale cellulære organeller for HCV-replikation, putativt tjener som virale samleanlæg 10, 11. I HCV-inficerede celler, det virale protein kerne og NS5A lokalisere til lipiddråber i en proces, der afhænger af triglycerid biosyntese, som inhibitorer af diacylglycerolacyltransferase-1 (DGAT1) forringe trafficking lipiddråber og efterfølgende HCV partikel produktion 12 </sup&gt;, 13, 14, 15. Desuden mutationer i lipid droplet-bindende domæner af enten kernen eller NS5A undertrykke HCV samling 16, 17. Kerne og NS5A derefter rekruttere alle andre virale proteiner, såvel som virale RNA replikationskomplekser, til membraner er tæt knyttet til lipid dråber 16. En samordnet indsats af alle virale proteiner er nødvendige for en vellykket produktion af infektiøs virusafkom 10, 11. De strukturelle proteiner er en del af virionerne, og de ikke-strukturelle proteiner fremme protein-protein-interaktioner, der kræves for denne proces. Interessant nok er det bona fide lipid dråbe protein PLIN3 / TIP47 kræves både HCV-RNA replikation og frigivelse af virioner 18, 19 <sup&gt;, 20. Trods disse nylige fremskridt, de mekanistiske detaljer, især af virus-værtssammenspil i de sene stadier af HCV-replikation, forbliver dårligt defineret, og den præcise funktion af lipiddråber er ukendt. Her beskriver vi en fremgangsmåde til isolering af lipiddråber til kvantitativ massespektrometri af associerede proteiner. Anvendelse af denne metode fandt vi dybtgående ændringer i lipid-dråben proteom under HCV-infektion og identificeret annexin A3 som en masse protein, som co-fraktionerer med lipiddråber og er nødvendig for effektiv HCV modning 21.

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			<td>SILAC Protein Quantitation Kit - DMEM</td>
			<td>Thermo Fisher</td>
			<td>89983&#160;</td>
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			<td>13C6 L-Arginine-HCl 50 mg</td>
			<td>Thermo Fisher</td>
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			<td>Roti-Load 1</td>
			<td>Roth GmbH</td>
			<td>K929.1</td>
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			<td>Roth GmbH</td>
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			<td>Geyer Th. GmbH &#38; Co.KG&#160;</td>
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			<td>GlutaMAX (100x)</td>
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			<td>Penicillin/Streptomycin Solution for Cell Culture</td>
			<td>Sigma-Aldrich Chemie GmbH&#160;</td>
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			<td>Sigma-Aldrich Chemie GmbH&#160;</td>
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			<td>Sigma-Aldrich Chemie GmbH&#160;</td>
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			<td>AppliChem GmbH</td>
			<td>A1149,1000</td>
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			<td>EDTA BioChemica</td>
			<td>AppliChem GmbH</td>
			<td>A1103,0250</td>
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			<td>Protease Inhibitor Cocktail 5ml</td>
			<td>Sigma-Aldrich Chemie GmbH&#160;</td>
			<td>P8340-5ML&#160;</td>
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			<td>D(+)-Sucrose BioChemica</td>
			<td>AppliChem GmbH</td>
			<td>A3935,1000</td>
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			<td>Hydrochloric acid 37% pure Ph. Eur., NF</td>
			<td>AppliChem GmbH</td>
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			<td>DC Protein Assay&#160;</td>
			<td>Bio-Rad Laboratoris GmbH&#160;</td>
			<td>500-0116&#160;</td>
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			<td>Glycerol</td>
			<td>AppliChem GmbH</td>
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			<td>AppliChem GmbH</td>
			<td>A1112</td>
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			<td>AppliChem GmbH</td>
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			<td>AppliChem GmbH</td>
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			<td>AppliChem GmbH</td>
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			<td>AppliChem GmbH</td>
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			<td>Potassium Phosphate Monobasic</td>
			<td>Sigma-Aldrich Chemie GmbH&#160;</td>
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			<td>Dipotassium Hydrogenphosphate</td>
			<td>Sigma-Aldrich Chemie GmbH&#160;</td>
			<td>P3786&#160;</td>
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			<td>DTT&#160;</td>
			<td>AppliChem GmbH</td>
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			<td>TWEEN 20</td>
			<td>AppliChem</td>
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			<td>Nonfat dried milk powder</td>
			<td>AppliChem</td>
			<td>A0830</td>
		</tr>
		<tr>
			<td>Anti-ADFP/ ADRP&#160;</td>
			<td>abcam</td>
			<td>ab52355</td>
		</tr>
		<tr>
			<td>M6PRB1/TIP47 100&#181;g&#160;</td>
			<td>abcam</td>
			<td>ab47639</td>
		</tr>
		<tr>
			<td>Calreticulin, pAb 200 &#181;g</td>
			<td>Enzo Life Science GmbH&#160;</td>
			<td>ADI-SPA-600-F&#160;</td>
		</tr>
		<tr>
			<td>Anti-&#223;-Tubulin&#160;</td>
			<td>Sigma-Aldrich Chemie GmbH&#160;</td>
			<td>T6074 200&#181;l &#160;</td>
		</tr>
		<tr>
			<td>Ethanol absolute</td>
			<td>Geyer Th. GmbH &#38; Co.KG&#160;</td>
			<td>A3678,0250 &#160;</td>
		</tr>
		<tr>
			<td>Anti-MnSOD</td>
			<td>Enzo Life Science GmbH&#160;</td>
			<td>ADI-SOD-110-F</td>
		</tr>
		<tr>
			<td>Anti-mouse HRP</td>
			<td>Thermo Fisher Pierce</td>
			<td>32430</td>
		</tr>
		<tr>
			<td>Anti-rabbit HRP</td>
			<td>Thermo Fisher&#160; Pierce</td>
			<td>32460</td>
		</tr>
		<tr>
			<td>Amersham Hyperfilm ECL</td>
			<td>GE Healthcare</td>
			<td>28906836</td>
		</tr>
		<tr>
			<td>Lumi-Light Western Blotting Substrate</td>
			<td>Sigma-Aldrich Chemie GmbH&#160;</td>
			<td>12015196001</td>
		</tr>
		<tr>
			<td>96 Well Cell Culture Plate</td>
			<td>Greiner Bio-One GmbH&#160;</td>
			<td>655 180&#160;</td>
		</tr>
		<tr>
			<td>Terumo Syringe 1 ml</td>
			<td>Terumo</td>
			<td>SS-01T</td>
		</tr>
		<tr>
			<td>Filtropur BT 50, 500 ml, 0.45 &#181;m&#160;</td>
			<td>SARSTEDT&#160;</td>
			<td>83.1823.100&#160;</td>
		</tr>
		<tr>
			<td>Mini-PROTEAN TGX Precast Gels, Any kD resolving gel</td>
			<td>Bio-Rad Laboratoris GmbH&#160;</td>
			<td>456-9034&#160;</td>
		</tr>
		<tr>
			<td>6 Well Cell Culture Plate</td>
			<td>Greiner Bio-One GmbH&#160;</td>
			<td>657160&#160;</td>
		</tr>
		<tr>
			<td>Dishes Nunclon 150/20&#160;</td>
			<td>Fisher Scientific GmbH&#160;</td>
			<td>10098720 - 168381&#160;</td>
		</tr>
		<tr>
			<td>Cell Scraper</td>
			<td>neoLab Migge GmbH&#160;</td>
			<td>C-8120&#160;</td>
		</tr>
		<tr>
			<td>Tube, 50 ml</td>
			<td>Greiner Bio-One GmbH&#160;</td>
			<td>227261&#160;</td>
		</tr>
		<tr>
			<td>SafeSeal Tube RNase-free&#160;</td>
			<td>SARSTEDT&#160;</td>
			<td>72.706.400&#160;</td>
		</tr>
		<tr>
			<td>Ultra Clear Centrifuge Tubes 11 x 60 mm</td>
			<td>Beckman Coulter GmbH&#160;</td>
			<td>344062&#160;</td>
		</tr>
		<tr>
			<td>Suction Needles</td>
			<td>Transcodent</td>
			<td>6482</td>
		</tr>
		<tr>
			<td>Biosphere Fil. Tip 1000&#160;</td>
			<td>SARSTEDT&#160;</td>
			<td>70.762.211&#160;</td>
		</tr>
		<tr>
			<td>Biosphere Fil. Tip 200</td>
			<td>SARSTEDT&#160;</td>
			<td>70.760.211&#160;</td>
		</tr>
		<tr>
			<td>Biosphere Fil. Tip 10</td>
			<td>SARSTEDT&#160;</td>
			<td>70.1130.210&#160;</td>
		</tr>
		<tr>
			<td>Dounce Tissue Grinder</td>
			<td>Fisher Scientific GmbH&#160;</td>
			<td>11883722</td>
		</tr>
		<tr>
			<td>Pestles For Dounce All-Glass Tissue Grinders</td>
			<td>Fisher Scientific GmbH&#160;</td>
			<td>10389444</td>
		</tr>
		<tr>
			<td>Orbitrap Fusion</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Branson Sonifier 450</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Thermomixer comfort, with Thermoblock 1.5 ml</td>
			<td>Eppendorf</td>
			<td>5355&#160;000.127</td>
		</tr>
		<tr>
			<td>Mini-PROTEAN Tetra Cell, Mini Trans-Blot Module, and PowerPac Basic Power Supply,&#160;</td>
			<td>BioRad</td>
			<td>165-8033</td>
		</tr>
		<tr>
			<td>Mini-PROTEAN 3 Multi-Casting Chamber</td>
			<td>BioRad</td>
			<td>165-4110</td>
		</tr>
		<tr>
			<td>PowerPac HC Power Supply</td>
			<td>Biorad</td>
			<td>164-5052</td>
		</tr>
		<tr>
			<td>Centrifuge&#160;</td>
			<td>Eppendorf</td>
			<td>5424R</td>
		</tr>
		<tr>
			<td>Centrifuge&#160;</td>
			<td>Eppendorf</td>
			<td>5424</td>
		</tr>
		<tr>
			<td>Optima L-90K</td>
			<td>Beckman Coulter GmbH&#160;</td>
			<td>365670</td>
		</tr>
		<tr>
			<td>SW 60 Ti Rotor</td>
			<td>Beckman Coulter GmbH&#160;</td>
			<td>335649</td>
		</tr>
		<tr>
			<td>Infinite M1000 PRO</td>
			<td>Tecan</td>
			<td></td>
		</tr>
	</tbody>
</table>

lipid droplet isolation for quantitative mass spectrometry analysis

Lipiddråber er afgørende for replikation af en række forskellige patogener, mest fremtrædende hepatitis C-virus (HCV), som det formodede site af virionmorfogenese. Kvantitativ lipid dråbe proteomanalyse kan anvendes til at identificere proteiner der lokaliserer til eller er forskudt fra lipiddråber under betingelser såsom virusinfektioner. Her beskriver vi en protokol, der er blevet brugt til at karakterisere ændringer i lipid dråbe proteom efter infektion med HCV. Vi bruger Stable Isotope Mærkning med aminosyrer i cellekultur (SILAC) og således mærke det samlede proteom af en population af celler med &quot;tunge&quot; aminosyrer til kvantificere proteiner ved massespektrometri. For lipid dråbe isolation er de to cellepopulationer (dvs. HCV-inficerede / &quot;light&quot; aminosyrer og ikke-inficerede kontrol / &quot;tunge&quot; aminosyrer) blandet 1: 1 og lyseret mekanisk i hypotonisk buffer. Efter fjernelse af kerner og celle debris ved lavhastighedscentrifugering, er lipid droplet-associerede proteiner beriget ved to efterfølgende ultracentrifugering trin efterfulgt af tre vasketrin i isotonisk puffer. Renheden af ​​lipid droplet fraktionerne analyseres ved Western blotting med antistoffer, der genkender forskellige subcellulære afsnit. Lipid dråbe-associerede proteiner adskilles derefter ved SDS-polyacrylamidgelelektroforese (SDS-PAGE) efterfulgt af Coomassie-farvning. Efter trypsinspaltningen peptiderne kvantificeret ved væskekromatografi-elektrosprayionisering-tandem massespektrometri (LC-ESI-MS / MS). Anvendelse af denne fremgangsmåde identificerede vi proteiner rekrutteret til lipiddråber upon HCV-infektion, der kan tyde pro- eller antivirale værtsfaktorer. Vores metode kan anvendes på en række forskellige celler og dyrkningsbetingelser, såsom infektion med patogener, miljømæssig stress, eller lægemiddelbehandling.

Lipiddråber er afgørende for HCV-infektion som de formodede steder for virionsamling, men de molekylære mekanismer i morfogenese og udstrømning af virioner er stort set ukendte. At identificere nye vært afhængighed faktorer involveret i den proces, udførte vi kvantitativ lipid dråbe proteomanalyse af HCV-inficerede celler 21 (figur 1A). Vi har etableret en protokol til oprensning af lipiddråber og rutinemæssigt detekteret en stærk berig...

Watch this Scientific Journal Video about Lipid Droplet Isolation for Quantitative Mass Spectrometry Analysis at JoVE.com

Lipid Droplet Isolation for Quantitative Mass Spectrometry Analysis

Lipiddråber er vigtige organeller for replikationen af ​​flere patogener, herunder hepatitis C-virus (HCV). Vi beskriver en fremgangsmåde til isolering af lipiddråber til kvantitativ massespektrometri af associerede proteiner; den kan anvendes under forskellige betingelser, såsom virusinfektion, miljømæssig stress, eller lægemiddelbehandling.

Lipid Droplet Isolation for Kvantitativ massespektrometri Analyse

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 ...

Research

JoVE Journal

Biochemistry

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

Time-løst elektrosprayioniserings Brint-deuterium Exchange-massespektrometri til at studere proteiners struktur og Dynamics

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling

Løsning affinitetsrensede proteinkomplekser ved Blå Native PAGE og protein Korrelation Profilering

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological ...

A Tandem Liquid Chromatography&#8211;Mass Spectrometry-based Approach for Metabolite Analysis of Staphylococcus aureus

A Tandem Liquid ChromatographyÃƒÆ’Ã‚Â¢ÃƒÂ¢Ã¢â‚¬Å¡Ã‚Â¬ÃƒÂ¢Ã¢â€šÂ¬Ã…â€œMass Spectrometry-based Approach for Metabolite Analysis of Staphylococcus aureus

En Tandem væskekromatografi-massespektrometri-baseret tilgang til Metabolit Analyse af<em&gt; Staphylococcus aureus</em

In an effort to thwart bacterial pathogens, hosts often limit the availability of nutrients at the site of infection. This limitation can alter the ...

Glycoproteomics of the Extracellular Matrix: A Method for Intact Glycopeptide Analysis Using Mass Spectrometry

Glycoproteomics af den ekstracellulære matrix: En metode til Intakt glycopeptid analyse ved hjælp massespektrometri

Fibrosis is a hallmark of many cardiovascular diseases and is associated with the exacerbated secretion and deposition of the extracellular matrix (ECM). ...

Proofreading and DNA Repair Assay Using Single Nucleotide Extension and MALDI-TOF Mass Spectrometry Analysis

Korrekturlæsning og DNA reparation analyse ved hjælp af Single Nucleotide udvidelse og MALDI-TOF-massespektrometri analyse

The maintenance of the genome and its faithful replication is paramount for conserving genetic information. To assess high fidelity replication, we have ...

Nitropeptide Profiling and Identification Illustrated by Angiotensin II

Nitropeptid profilering og identifikation illustreret af angiotensin II

Protein nitration is one of the most important post-translational modifications (PTM) on tyrosine residues and it can be induced by chemical actions of ...

Sample Preparation for Probe Electrospray Ionization Mass Spectrometry

Prøve forberedelse til Sonde Elektrospray Ionization Mass Spektrometri

Mass spectrometry (MS) is a powerful tool in analytical chemistry because it provides very accurate information about molecules, such as mass-to-charge ...

Use of Microscale Thermophoresis to Measure Protein-Lipid Interactions

Anvendelse af mikroskala termoforese til måling af protein-lipid interaktioner

The ability to determine the binding affinity of lipids to proteins is an essential part of understanding protein-lipid interactions in membrane ...

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

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

Kvantitativ analyse af cellulære lipidom af Saccharomyces Cerevisiae Ved hjælp af flydende kromatografi kombineret med tandem massespektrometri

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

Untargeted Liquid Chromatography-Mass Spectrometry-Based Metabolomics Analysis of Wheat Grain

Umålrettet flydende kromatografi-massespektrometri-baserede metabolomiske analyse af hvedekorn

Understanding the interactions between genes, the environment and management in agricultural practice could allow more accurate prediction and management ...