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

Dit werk werd ondersteund door een ARRA aanvulling op de levensduur / Tufts / Brown CFAR, P30AI042853-13S1, NIH P20GM103421, P01AA019072, R01HD072693 en K24HD080539 tot BR. Dit werk werd ook gesteund door Levensduur Pilot Research Fund (# 701- 5857), Rhode Island Stichting Medical Research Grant (# 20.133.969), en NIH COBRE URI / RIH Pilot Research Grant (P20GM104317) naar ML. Wij danken James Myall en Vy Dang voor hulp bij het manuscript en figuur voorbereiding.

1. Celkweek en HIV-1 infectie LET OP: Voordat u begint met experimenten, is het raadzaam om de levensvatbaarheid van de cellen door middel van trypan blauwe vlekken 11 en de verspreiding daarvan te controleren door middel van een MTT-test 12. Het is ook van cruciaal belang voor nieuw bereide SILAC medium te gebruiken. Verschillende cellijnen kunnen worden gebruikt, mits ze in een actief proliferatieve fase en zijn gevoelig voor HIV-1 infectie, of...

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In de in dit artikel beschreven procedures, we aangetoond dat de toepassing van de SILAC techniek om het effect van HIV-1 infectie bij de gastheer exosomaal proteoom onderzoeken. Aanvankelijk geïnfecteerde en HIV-1 geïnfecteerde cellen verschillend isotoop gemerkte. De verschillend gemerkte exosomes worden vervolgens gezuiverd voordat eiwitextractie. Vervolgens wordt vloeistofchromatografie-tandem massaspectrometrie gebruikt om de exosomaal proteoom analyse. Ten slotte is de resulterende massa spectrometrie gegevens e...

Veel menselijke ziekten, waaronder virusinfecties, vaak geassocieerd met kenmerkende cellulaire processen die plaatsvinden in en rond de aangetaste cellen. Eiwitten, die vaak optreedt als de ultieme mobiele effectoren, bemiddelen deze processen. Analyse van de eiwitten vaak waardevolle informatie verschaffen over de lokale omgeving van betrokken cellen en helpen om het onderliggende mechanisme van de pathogenese van de ziekte te begrijpen. Tussen de verschillende eiwitanalyse technieken, proteomics houdt bijzonder grote belofte. Als krachtige, grootschalige gereedschap kan proteomics systemische begrip van cellulaire processen, met name op het gebied van de functie en interactie van eiwitten. Analyseren eiwitten wordt eenvoudiger gemaakt door de ontwikkeling van labeling technieken, waarmee onderzoekers om de expressie van cellulaire componenten, met name eiwitten controleren, in de plaats van onderzoek. Hoewel veel proteomics analyses zijn uitgevoerd op cellulairproteoom schaal, proteomics karakteriseringen op subcellulaire compartimenten bleek bijzonder informatief 1 te zijn. Dit wordt goed geïllustreerd in de studies van HIV-1 infectie. Exosomes, 30-100 nm membraanblaasjes afgescheiden door een groot aantal celtypen 2, 3, zijn kritische componenten van intercellulaire communicatie en moleculair transport. Ze werden eerder ontdekte belangrijke rol spelen in de HIV-1 ontluikende werkwijze 4, 5. Door het combineren proteoomanalyse met functionele dissectie, vonden we dat exosomes vrijkomt uit HIV-1 geïnfecteerde cellen bestaan uit een unieke en kwantitatief andere handtekening en haven eiwit regulerende moleculen die cellulaire eigenschappen beïnvloeden naburige ontvankelijke cellen, inclusief cellulaire apoptose en proliferatie 6. De methoden worden beschreven in dit protocol,namelijk SILAC (stabiele isotoop labeling van aminozuren in celcultuur) 7 op basis van proteomics karakterisering van exosomes van HIV-1 geïnfecteerde cellen. Soortgelijke benaderingen kunnen worden toegepast om beter inzicht andere subcellulaire compartimenten tijdens pathogenese door het aanpassen van de experimentele belasting van de specifieke compartiment of gedeelte plaats en een aantal wijzigingen aan de beschreven procedures. Gezien de recente ontwikkeling van kwantitatieve proteomics methoden, zijn er veel te kiezen bij het selecteren van de meest efficiënte methode voor een bepaald experiment. Voorbeelden hiervan zijn de chemische basis iTRAQ (isobarisch tags voor relatieve en absolute kwantificatie) 8 en het label-free MRM (meerdere reactie monitoring) 9 technieken. Beide methoden zijn krachtige tools en zijn goede keuzes voor specifieke instellingen. Voor een typische laboratorium vooral bezig met cellijnen, maar deze twee methoden relatively hogere kosten en meer tijdrovend in vergelijking met de SILAC gebaseerde methode. SILAC is een metabole gebaseerde labeling techniek die radioactieve isotopische vormen van aminozuren uit het kweekmedium in cellulaire eiwitten bevat. Gewoonlijk SILAC experimenten beginnen met twee celpopulaties, bijvoorbeeld geïnfecteerde en ongeïnfecteerde. Elke kamer is verschillend gemerkte in zijn specifieke isotoop omgeving tot volledige etikettering is bereikt. De gelabelde exosomes van deze cellen worden vervolgens onderworpen aan proteïne extractie. Eenmaal geëxtraheerd, de gelabelde exosomaal eiwitten worden geanalyseerd met vloeistofchromatografie tandem 10 massaspectroscopie. Tenslotte de massaspectrometrie resultaten en significant gemerkte eiwitten worden onderworpen aan statistische en bioinformatica analyses en biochemische strenge controle. Onze eerdere onderzoeksrapporten suggereren dat de SILAC / exosome procedures geschikter voor cellijnen van primaire cellen, cellijnen zijn gewoonlijk peractief prolifererende staat voor een efficiënte isotopische labeling,

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		<tr height="21">
			<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.

Figuur 1A is een stroomkaart die de werkwijze 21 SILAC labeling. Om de exosomes zuiveren, moet de monsters te worden gesponnen via centrifuge. Figuur 1B toont de stappen van exosome zuivering door ultracentrifugatie seriële 21. Eenmaal gezuiverd, de exosomes onderworpen aan experimentele proteoomanalyse zoals bepaald in de procedure. Fig...

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

We beschrijven een kwantitatieve proteomics met gebruik van technieken van stabiele isotopen van aminozuren in celcultuur (SILAC) het effect van HIV-1 infectie op host exosomaal proteoom analyse. Dit protocol kan gemakkelijk worden aangepast aan cellen onder verschillende stress of infectie omstandigheden.

SILAC Based Proteoom karakterisatie van exosomen van HIV-1 geïnfecteerde cellen

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

Specifieke markering van de HIV-1 positieve cellen met behulp van een Rev-afhankelijke lentivirale vector uitdrukken van de 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

Genotypische Inferentie van HIV-1 tropisme de populatie-gebaseerde Sequencing van 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

Bereiding en het gebruik van HIV-1 geïnfecteerde primaire CD4 + T-cellen als doelcellen in Natural Killer Cell Cytotoxische 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

Versterken en kwantificeren van HIV-1 RNA in HIV-geïnfecteerde personen met een viral load onder de limiet van de Opsporing van 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 van HIV-1 Kernen

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

Nieuwe tools om regulatoire T-cellen van HIV-1-geïnfecteerde individuen Expand

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.

Het beoordelen van de Aangeboren Sensing van HIV-1 geïnfecteerde CD4<sup&gt; +</sup&gt; T cellen door plasmacytoïde dendritische cellen gebruiken een<em&gt; Ex vivo</em&gt; Co-cultuur systeem.

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

Conformationele Evaluatie van HIV-1 Trimere envelop glycoproteïnen met een cel-gebaseerde ELISA test

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

Isolatie van exosomen uit het plasma van HIV-1 positieve personen

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. ...