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

Detta arbete stöddes av ett ARRA tillägg till Livslängd / Tufts / brun CFAR, P30AI042853-13S1, NIH P20GM103421, P01AA019072, R01HD072693 och K24HD080539 till BR. Detta arbete stöddes också av Lifespan Pilot Research Fund (# 701 5857), Rhode Island Foundation Medical Research Grant (# 20.133.969) och NIH COBRE URI / RIH Pilot bidrag (P20GM104317) till ML. Vi tackar James Myall och Vy Dang för hjälp med manuskriptet och figur förberedelse.

1. Cell Culture och HIV-1-infektion OBS: Innan experiment, är det rekommenderat att kontrollera cellernas livsduglighet genom Trypan blå färgning 11 och deras spridning genom en MTT-analys 12. Det är också viktigt att använda nyligen preparerat SILAC medium. Olika cellinjer kan användas, så länge som de är i en aktivt proliferativt stadium, och är mottagliga för HIV-1-infektion, eller testvillkoret av valet. I detta protokoll använde...

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I de förfaranden som beskrivs i detta dokument visade vi tillämpningen av SILAC teknik för att undersöka effekten av HIV-1-infektion på värd exosomal proteom. Initialt, oinfekterade och HIV-1-infekterade celler är differentiellt isotopmärkt. De differentiellt märkta exosomes renas sedan innan du utför proteinextraktion. Därefter vätskekromatografi-tandem-masspektrometri användes för att analysera exosomal proteom. Slutligen, de resulterande masspektrometri data och potentiella kandidatproteiner utsattes f?...

Många mänskliga sjukdomar, inklusive virusinfektioner, är ofta förknippade med distinkta cellulära processer som äger rum i och runt de drabbade cellerna. Proteiner, ofta i egenskap av den ultimata cellulära effektorer, medla dessa processer. Analys av proteinerna ofta kan ge ovärderlig information om den lokala miljön av påverkade celler och hjälpa oss att förstå den bakomliggande mekanismen för sjukdom patogenes. Bland olika proteinanalystekniker, har proteomik särskilt stort löfte. Som ett kraftfullt, storskaliga verktyg kan proteomik tillhandahålla en systemisk förståelse av cellulära processer, i synnerhet i området av funktionen och interaktionen av proteiner. Analysera specifika proteiner görs enklare genom utveckling av märkningstekniker, som tillåter forskare att övervaka uttrycket av cellulära komponenter, i synnerhet proteiner, i stället för undersökningen. Även om många proteomik analyser har gjorts på cellproteom skala, proteomik karakteriseringar på subcellulära fack har visat sig vara särskilt upplysande en. Detta exemplifieras väl i studier av HIV-1-infektion. Exosomes, 30-100 nm membranvesiklar som utsöndras av ett brett spektrum av celltyper 2, 3, är kritiska komponenter i intercellulär kommunikation och molekylär transport. De tidigare upptäckt att spela viktiga roller i HIV-1 spirande process 4, 5. Genom att kombinera proteomik analys med funktionell dissekering, fann vi att exosomes frigörs från HIV-1-infekterade celler består av en unik och kvantitativt olika protein signatur och hamnen reglerande molekyler som påverkar cellulära egenskaper på angränsande receptiva celler, inklusive cellulär apoptos och proliferation 6. Metoderna beskrivs i detta protokoll,nämligen SILAC (stabil isotop märkning av aminosyror i cellkultur) 7 baserat proteomic karakterisering av exosomer från HIV-1-infekterade celler. Liknande tillvägagångssätt kan användas för att bättre förstå andra subcellulära fack under patogenes genom att justera experimentell spännings till den specifika avdelning eller del av intresse och göra nödvändiga ändringar av de beskrivna förfarandena. Med tanke på den senaste utvecklingen av kvantitativa proteomik metoder, det finns många att välja mellan när du väljer den mest effektiva metoden för en viss experiment. Bland dessa är den kemiska-baserade iTRAQ (isobariska taggar för relativ och absolut kvantifiering) 8 och etiketten fria MRM (multipel övervakning reaktion) 9 tekniker. Båda metoderna är kraftfulla verktyg och är bra val för specifika inställningar. För en typisk laboratorie huvudsakligen arbeta med cellinjer, emellertid är dessa två metoder har relatively högre kostnader och är mer tidskrävande i jämförelse med den SILAC baserade metoden. SILAC är en metabolisk baserat märkningsteknik som innehåller icke-radioaktiva isotopiska former av aminosyror från odlingsmedierna i cellulära proteiner. Typiskt SILAC experiment börjar med två cellpopulationer, till exempel, infekterade och oinfekterade. Varje differentiellt märkt i sin specifika isotop miljö tills full märkning uppnås. De märkta exosomer av dessa celler utsätts sedan för proteinextraktion. När utvunnits, de märkta exosomal proteinerna analyseras med hjälp av vätskekromatografi tandem masspektroskopi 10. Slutligen masspektrometri resultat och kraftigt märkta proteiner utsattes för statistisk och bioinformatik analyser samt strikt biokemisk kontroll. Våra tidigare undersökande rapporter tyder på att SILAC / Exosome förfaranden är mer lämpade för cellinjer än primära celler, som cellinjer är vanligtvis i enaktiv prolifererande tillstånd för effektiv isotopmärkning,

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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 är ett flödesschema som beskriver förfarandet 21 den SILAC märkning. För att rena exosomes måste proverna centrifugeras ned via centrifug. Figur 1B visar stegen Exosome rening genom serieultracentrifugering 21. När renad, de exosomes är föremål för experimentell proteomik analys som beskrivs i förfarandet. Figur 2A</s...

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

Här beskriver vi en kvantitativ proteomik metod som använder tekniken med stabila isotopen märkning av aminosyror i cellkultur (SILAC) att analysera effekterna av HIV-1-infektion på värd exosomal proteom. Detta protokoll kan enkelt anpassas till celler under olika stress eller infektionsförhållanden.

SILAC Based proteomik karakterisering av exosomes från HIV-1-infekterade 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

Särskild märkning av HIV-1 positiva celler med hjälp av en Rev-beroende Lentiviral Vector uttrycka det grönt fluorescerande proteinet

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

Genotypisk Inferens av HIV-1 Tropism Använda Befolkning-sekvensering av 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

Beredning och användning av HIV-1 infekterade Primär CD4 + T-celler som målceller i Natural Cytotoxiska Killer Cell analyser

 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

Förstärka och kvantifiera HIV-1 RNA i hiv-infekterade individer med virusnivåer under gränsen för upptäckt med vanliga kliniska analyser

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 av HIV-1 kärnor

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

Nya verktyg för att expandera reglerande T-celler från HIV-1-infekterade individer

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.

Bedömning av Innate Sensing av HIV-1 infekterade CD4<sup&gt; +</sup&gt; T-celler från Plasmacytoid dendritiska celler med en<em&gt; Ex vivo</em&gt; Samodlingssystemet.

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

Conformational Utvärdering av HIV-1 trimera Envelope Glycoproteins användning av en cellbaserad ELISA-analys

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 av exosomes från plasma HIV-1 positiva individer

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. ...