Name: silac based proteomic characterization of exosomes from hiv-1 infected cells
Uploaded: 2017-03-03T12:30:00
Description: Watch this Scientific Journal Video about SILAC Based Proteomic Characterization of Exosomes from HIV-1 Infected Cells at JoVE.com

Dette arbejde blev støttet af en arra supplement til Levetid / Tufts / Brown CFAR, P30AI042853-13S1, NIH P20GM103421, P01AA019072, R01HD072693, og K24HD080539 til BR. Dette arbejde blev også støttet af Levetid Research Fund Pilot (# 701- 5857), Rhode Island Foundation Medical Research Grant (# 20.133.969), og NIH COBRE URI / RIH Pilot Research Grant (P20GM104317) til ML. Vi takker James Myall og Vy Dang efter hjælp til manuskriptet og figur forberedelse.

1. Cell Culture og HIV-1-infektion BEMÆRK: Før du starter eksperimenter, anbefales det at kontrollere cellernes levedygtighed gennem Trypan blå farvning 11 og deres spredning gennem en MTT assay 12. Det er også vigtigt at bruge frisklavet SILAC medium. Forskellige cellelinier kan anvendes, så længe de er i en aktivt proliferative fase, og er modtagelige for HIV-1-infektion, eller test valgte tilstand. I denne protokol, bruge H9 cellelinje s...

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I procedurerne i dette papir, demonstrerede vi anvendelsen af ​​SILAC teknik til at undersøge effekten af ​​HIV-1 infektion på værten exosomal proteomanalyse. Oprindeligt uinficerede og HIV-1-inficerede celler er differentielt isotopmærket. De differentielt mærkede exosomer renses derefter før udførelse protein ekstraktion. Dernæst væskekromatografi-tandem-massespektrometri anvendt til at analysere exosomal proteom. Endelig er de resulterende massespektrometri data og potentielle kandidatlande proteiner...

Mange humane sygdomme, herunder virale infektioner, er ofte forbundet med karakteristiske cellulære processer, der finder sted i og omkring de ramte celler. Proteiner, der ofte fungerer som den ultimative cellulære effektorer, mægle disse processer. Analyse af proteinerne ofte kan give uvurderlig oplysninger om det lokale miljø af berørte celler og hjælpe os til at forstå den underliggende mekanisme af sygdom patogenese. Blandt de forskellige protein analyseteknikker, proteomics holder særligt meget lovende. Som en stærk, storstilet værktøj, kan proteomics tilvejebringe en systemisk forståelse af cellulære processer, især med hensyn til funktion og interaktion af proteiner. Analysere specifikke proteiner er blevet forenklet gennem udvikling af mærkningsteknikker, som giver forskere for at overvåge ekspressionen af ​​cellulære bestanddele, især proteiner, i stedet for undersøgelsen. Selv om mange proteomikanalyser er blevet udført på celleniveauproteom skala, proteomiske karakteriseringer på subcellulære afsnit har vist sig at være særligt informativ 1. Dette er eksemplificeret godt i undersøgelser af HIV-1-infektion. Exosomer, 30-100 nm membranvesikler udskilles af en lang række celletyper 2, 3, er kritiske komponenter i intercellulær kommunikation og molekylær transport. De blev tidligere opdaget at spille vigtige roller i HIV-1 celleafsnørringsproces 4, 5. Ved at kombinere proteomisk analyse med funktionelle dissektion, fandt vi, at exosomer frigivet fra HIV-1-inficerede celler er sammensat af en unik og kvantitativt forskellige protein signatur og havnen regulatoriske molekyler, der påvirker cellulære ejendomme i nabolandet receptive celler, herunder cellulær apoptose og proliferation 6. Metoderne er beskrevet i denne protokol,nemlig SILAC (stabil isotop mærkning af aminosyrer i cellekultur) 7 baseret proteomisk karakterisering af exosomer fra HIV-1-inficerede celler. kan anvendes lignende metoder til bedre at forstå andre subcellulære rum under patogenese ved at justere den eksperimentelle stress til den specifikke rum eller brøkdel af interesse og foretage de nødvendige ændringer af de beskrevne procedurer. I betragtning af den seneste udvikling af kvantitative proteom metoder, der er mange at vælge imellem, når du vælger den mest effektive metode til et bestemt eksperiment. Blandt disse er den kemisk-baserede iTRAQ (isobare tags for relativ og absolut kvantificering) 8 og etiketten fri MRM (multiple overvågning reaktion) 9 teknikker. Begge metoder er effektive værktøjer og er gode valg for specifikke indstillinger. For en typisk laboratorium arbejder hovedsageligt med cellelinier, men disse to metoder har relatively højere omkostninger og er mere tidskrævende sammenlignet med den SILAC metode. SILAC er en metabolisk baseret mærkning teknik, der inkorporerer ikke-radioaktive isotopiske former af aminosyrer fra dyrkningsmediet i cellulære proteiner. Typisk SILAC eksperimenter starter med to cellepopulationer, for eksempel, inficerede og ikke-inficerede. Hver er forskelligt mærket i sin specifik isotopisk miljø indtil fuld mærkning er opnået. De mærkede exosomer af disse celler underkastes derefter proteinekstraktion. Når ekstraheret, de mærkede exosomal proteiner analyseres ved anvendelse væskekromatografi tandem massespektroskopi 10. Endelig er massespektrometri resultater og betydeligt mærkede proteiner underkastet statistisk og bioinformatik analyser samt streng biokemisk verifikation. Vores tidligere efterforskningsmæssige rapporter antyder, at de SILAC / exosom procedurer er mere passende for cellelinier end primære celler, som cellelinier er normalt i enaktiv prolifererende tilstand til effektiv isotopisk mærkning,

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			<td height="21" style="height:21px;width:227px;">H9 cell line</td>
			<td style="width:121px;">ATCC</td>
			<td style="width:111px;">HTB-176</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Trypan Blue</td>
			<td style="width:121px;">Thermo Fisher</td>
			<td style="width:111px;">15250061</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">MTT assay kit</td>
			<td style="width:121px;">Thermo Fisher</td>
			<td style="width:111px;">V13154</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:26px;width:227px;">Dialyzed fetal bovine serum (FBS)</td>
			<td style="width:121px;">Thermo Fisher</td>
			<td style="width:111px;">26400044</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">SILAC Protein Quantitation Kit &#8211; RPMI 1640</td>
			<td style="width:121px;">Thermo Fisher</td>
			<td style="width:111px;">89982</td>
			<td style="width:168px;">DMEM version (89983)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">L-Arginine-HCl, 13C6, 15N4 for SILAC</td>
			<td style="width:121px;">Thermo Fisher</td>
			<td style="width:111px;">88434</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">L-Lysine-2HCl, 13C6 for SILAC</td>
			<td style="width:121px;">Thermo Fisher</td>
			<td style="width:111px;">88431</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">HIV-1NL4-3&#160;&#160;</td>
			<td style="width:121px;">NIH AIDS Reagent Program</td>
			<td style="width:111px;">2480</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">Alliance HIV-1 p24 Antigen ELISA kit</td>
			<td style="width:121px;">PerkinElmer</td>
			<td style="width:111px;">NEK050001KT</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">Refrigerated super-speed centrifuge</td>
			<td style="width:121px;">Eppendorf</td>
			<td style="width:111px;">22628045</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">Refrigerated ultracentrifuge</td>
			<td style="width:121px;">Beckman Coulter</td>
			<td style="width:111px;">363118</td>
			<td style="width:168px;">Should be able to reach 100,000g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">50mL Conical Centrifuge Tubes</td>
			<td style="width:121px;">Thermo Fisher</td>
			<td style="width:111px;">14-432-22</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Ultracentrifuge Tubes</td>
			<td style="width:121px;">Beckman Coulter</td>
			<td style="width:111px;">326823</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">SW 32 Ti Rotor</td>
			<td style="width:121px;">Beckman Coulter</td>
			<td style="width:111px;">369694</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">RIPA buffer</td>
			<td style="width:121px;">Thermo Fisher</td>
			<td style="width:111px;">89900</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Protease Inhibitor Cocktails</td>
			<td style="width:121px;">Thermo Fisher</td>
			<td style="width:111px;">&#160;78430</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">ThermoMixer&#160;</td>
			<td style="width:121px;">Eppendorf</td>
			<td style="width:111px;">5384000020</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">BCA Protein Assay Kit&#160;</td>
			<td style="width:121px;">Thermo Fisher</td>
			<td style="width:111px;">23250</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Spectrophotometer</td>
			<td style="width:121px;">Biorad</td>
			<td style="width:111px;">1702525</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">SDS PAGE Gel apparatus</td>
			<td style="width:121px;">Thermo Fisher</td>
			<td style="width:111px;">EI0001</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">Novex 4-20% Tris-Glycine Mini Gels</td>
			<td style="width:121px;">Novex</td>
			<td style="width:111px;">XV04200PK20</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Gel staining reagent</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td style="width:111px;">G1041</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">Sequencing Grade Modified Trypsin</td>
			<td style="width:121px;">Promega</td>
			<td style="width:111px;">V5111</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">SpeedVac Concentrator</td>
			<td style="width:121px;">Thermo Fisher</td>
			<td style="width:111px;">SPD131DDA</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Antibody to human annexin A5</td>
			<td style="width:121px;">Abcam</td>
			<td style="width:111px;">ab14196</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">Antibody to human lactate dehydrogenase B chain</td>
			<td style="width:121px;">Abcam</td>
			<td style="width:111px;">ab53292</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:227px;">Graphing and Statistical Software</td>
			<td style="width:121px;">Systat&#160;</td>
			<td style="width:111px;">SigmaPlot&#160;</td>
			<td style="width:168px;">Or GraphPad Prism</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:227px;">Quantitative proteomics software suite</td>
			<td style="width:121px;">Max Planck Institue of Biochemistry</td>
			<td style="width:111px;">Maxquant&#160;</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">Software and databases</td>
			<td style="width:121px;">Various vendors</td>
			<td style="width:111px;"></td>
			<td style="width:168px;">Refer to main text for details</td>
		</tr>
	</tbody>
</table>

silac based proteomic characterization of exosomes from hiv-1 infected cells

Proteomics is the large-scale analysis of proteins. Proteomic techniques, such as liquid chromatography tandem mass spectroscopy (LC-MS/MS), can characterize thousands of proteins at a time. These powerful techniques allow us to have a systemic understanding of cellular changes, especially when cells are subjected to various stimuli, such as infections, stresses, and specific test conditions. Even with recent developments, analyzing the exosomal proteome is time-consuming and often involves complex methodologies. In addition, the resultant large dataset often needs robust and streamlined analysis in order for researchers to perform further downstream studies. Here, we describe a SILAC-based protocol for characterizing the exosomal proteome when cells are infected with HIV-1. The method is based on simple isotope labeling, isolation of exosomes from differentially labeled cells, and mass spectrometry analysis. This is followed by detailed data mining and bioinformatics analysis of the proteomic hits. The resultant datasets and candidates are easy to understand and often offer a wealth of information that is useful for downstream analysis. This protocol is applicable to other subcellular compartments and a wide range of test conditions.

Figur 1A er et rutediagram der skitserer SILAC mærkningsproceduren 21. For at oprense exosomer, skal prøven centrifugeret ned via centrifuge. Figur 1B viser trinnene exosome Oprensning ved seriel ultracentrifugering 21. Når oprenset, exosomerne underkastes eksperimentel proteom analyse som skitseret i proceduren. Figur 2A er e...

Watch this Scientific Journal Video about SILAC Based Proteomic Characterization of Exosomes from HIV-1 Infected Cells at JoVE.com

SILAC Based Proteomic Characterization of Exosomes from HIV-1 Infected Cells

Her beskriver vi en kvantitativ proteomics metode ved hjælp af teknikken af ​​stabile isotoper mærkning af aminosyrer i cellekultur (SILAC) at analysere virkningerne af HIV-1 infektion på host exosomal proteomer. Denne protokol kan let tilpasses til celler under forskellige stress eller infektion betingelser.

SILAC Based proteomiske Karakterisering af Exosomer fra HIV-1 inficerede celler

Proteomics is the large-scale analysis of proteins. Proteomic techniques, such as liquid chromatography tandem mass spectroscopy (LC-MS/MS), can ...

Research

JoVE Journal

Immunology and Infection

Specific Marking of HIV-1 Positive Cells using a Rev-dependent Lentiviral Vector Expressing the Green Fluorescent Protein

Most of HIV-responsive expression vectors are based on the HIV promoter, the long terminal repeat (LTR). While responsive to an early HIV protein, Tat, ...

Genotypic Inference of HIV-1 Tropism Using Population-based Sequencing of V3

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her ...

Preparation and Use of HIV-1 Infected Primary CD4+ T-Cells as Target Cells in Natural Killer Cell Cytotoxic Assays

 Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK ...

Amplifying and Quantifying HIV-1 RNA in HIV Infected Individuals with Viral Loads Below the Limit of Detection by Standard Clinical Assays

Amplifying viral genes and quantifying HIV-1 RNA in HIV-1 infected individuals with viral loads below the limit of detection by standard assays (below ...

In vitro Uncoating of HIV-1 Cores

In vitro Uncoating of HIV-1 Cores

In vitro Uncoating af HIV-1 kerner

The genome of the retroviruses is encased in a capsid surrounded by a lipid envelope. For lentiviruses, such as HIV-1, the conical capsid shell is ...

New Tools to Expand Regulatory T Cells from HIV-1-infected Individuals

Nye værktøjer til at udvide regulatoriske T celler fra HIV-1-inficerede personer

CD4+ Regulatory T cells (Tregs) are potent immune modulators and serve an important function in human immune homeostasis. Depletion of Tregs has led to ...

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

Vurdering af Innate Sensing af HIV-1 inficerede CD4<sup&gt; +</sup&gt; T-celler ved plasmacytoide dendritiske celler ved hjælp af en<em&gt; Ex vivo</em&gt; Co-dyrkningssystem.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the ...

Conformational Evaluation of HIV-1 Trimeric Envelope Glycoproteins Using a Cell-based ELISA Assay

Konformationel Evaluering af HIV-1 trimere kappeglycoproteinerne hjælp af en celle-ELISA-assay

HIV-1 envelope glycoproteins (Env) mediate viral entry into target cells and are essential to the infectious cycle. Understanding how those glycoproteins ...

Isolation of Exosomes from the Plasma of HIV-1 Positive Individuals

Isolering af Exosomer fra plasma af HIV-1 Positive Personer

Exosomes are small vesicles ranging in size from 30 nm to 100 nm that are released both constitutively and upon stimulation from a variety of cell types. ...