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 arbeidet ble støttet av en Arra supplement til Livsløp / Tufts / Brown CFAR, P30AI042853-13S1, NIH P20GM103421, P01AA019072, R01HD072693, og K24HD080539 til BR. Dette arbeidet ble også støttet av Levetid Pilot forskningsfond (# 701- 5857), Rhode Island Foundation Medical Research Grant (# 20133969) og NIH Cobre URI / RIH Pilot stipend (P20GM104317) til ML. Vi takker James Myall og Vy Dang for hjelp med manuskriptet og figur forberedelse.

1. Cellekultur og HIV-1-infeksjon MERK: Før du starter eksperimenter, anbefales det å sjekke cellenes levedyktighet gjennom trypanblått farging 11 og deres spredning gjennom en MTT-analyse 12. Det er også viktig å bruke nypreparerte SILAC medium. Forskjellige cellelinjer kan anvendes, så lenge de er i en aktiv proliferativ fase, og er utsatt for HIV-1-infeksjon, eller testbetingelse av valget. I denne protokollen, bruker H9 cellelinje som e...

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I fremgangsmåtene som beskrives i dette dokumentet, har vi demonstrert anvendelsen av SILAC teknikk for å undersøke effekten av HIV-1-infeksjon i verten exosomal proteome. I utgangspunktet friske og HIV-1-infiserte celler er forskjellig isotop-merket. De forskjellig merkede exosomes blir deretter renset før du utfører protein utvinning. Deretter blir væskekromatografi tandem-massespektrometri anvendes for å analysere exosomal proteome. Til slutt blir den resulterende masse spektrometri data og potensiell kandidat...

Mange menneskelige sykdommer, innbefattende virusinfeksjoner, er ofte forbundet med karakteristiske cellulære prosesser som finner sted i og rundt de berørte celler. Proteiner, ofte fungerer som den ultimate mobile effektbokser, megle disse prosessene. Analyse av proteiner ofte kan gi uvurderlig informasjon om det lokale miljøet av syke cellene og hjelpe oss til å forstå den underliggende mekanismen av sykdom patogenesen. Blant ulike protein analyseteknikker, proteomikk har særlig store løftet. Som et kraftig, stor-skala-verktøyet, kan proteomforskning tilveiebringe en systemisk forståelse av cellulære prosesser, spesielt i området av funksjon og interaksjonen av proteiner. Analysere spesifikke proteiner er gjort enklere gjennom utvikling av merkingsteknikker, som tillater forskere for å overvåke ekspresjon av cellulære komponenter, spesielt proteiner, i stedet for undersøkelsen. Selv om mange proteomikk analyser har blitt utført på celleproteom skala, proteomikk characterizations på subcellulære avdelinger har vist seg å være spesielt informativ en. Dette eksemplifiseres godt i studiene av HIV-1-infeksjon. Exosomes, 30-100 nm membranvesikler utskilt av et bredt spekter av celletyper 2, 3, er kritiske komponenter av intercellulær kommunikasjon og molekylære transport. De ble tidligere funnet å spille viktige roller i HIV-1 spirende prosess 4, 5. Ved å kombinere proteomikk analyse med funksjonelle disseksjon, fant vi at exosomes frigjøres fra HIV-1-infiserte celler består av en unik og kvantitativt forskjellige protein signatur og havne regulatoriske molekyler som påvirker celleegenskaper på nabo mottakelige celler, inkludert cellulær apoptose og proliferasjon 6. Metodene er beskrevet i denne protokoll,nemlig SILAC (stabil isotop merking av aminosyrer i cellekultur) 7 basert proteomikk karakterisering av exosomes fra HIV-1-infiserte celler. Lignende metoder kan anvendes for å bedre forstå andre subcellulære kamrene under patogenesen ved å justere den eksperimentelle spenning til den spesifikke rommet eller fraksjon av interesse, og å gjøre nødvendige endringer i de beskrevne fremgangsmåter. Gitt den siste utviklingen av kvantitative proteomikk metoder, er det mange å velge mellom når du velger den mest effektive metoden for et bestemt eksperiment. Blant disse er den kjemiske-baserte iTRAQ (isobariske koder for relativ og absolutt kvantifisering) 8 og etiketten frie MRM (multiple reaksjon overvåking) 9 teknikker. Begge metodene er kraftige verktøy, og er gode valg for bestemte innstillinger. For en typisk laboratorie hovedsakelig arbeider med cellelinjer, men disse to metodene har relatively høyere kostnader og er mer tidkrevende sammenlignet med den SILAC basert metode. SILAC er en metabolsk merking basert teknikk som omfatter ikke-radioaktive isotopiske former av aminosyrer fra kulturmediet til cellulære proteiner. Vanligvis SILAC eksperimenter starte med to cellepopulasjoner, for eksempel, infiserte og uinfiserte. Hver er forskjellig merket i sin bestemt isotop-miljøet før full merking er oppnådd. De merkede exosomes av disse cellene blir deretter utsatt for proteinekstraksjon. Når ekstraheres, de merkede exosomal proteiner blir analysert ved hjelp av væskekromatografi tandem massespektroskopi 10. Til slutt blir de massespektrometri resultater og betydelig merkede proteiner underkastet statistisk og bioinformatikk analyser, samt streng biokjemisk verifisering. Våre tidligere undersøkende rapporter tyder på at de SILAC / exosome prosedyrer er mer hensiktsmessig for cellelinjer enn primærceller, som cellelinjer er vanligvis i enaktiv prolifererende tilstand for effektiv isotopisk merking,

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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.

Figur 1A er et flytskjema som beskriver SILAC merking prosedyren 21. For å rense exosomes, må prøvene spunnet ned via sentrifuge. Figur 1B viser trinnene av exosome rensing av serie ultracentrifugation 21. Når renset, er exosomes er gjenstand for eksperimentell proteomikk analyse som beskrevet i prosedyren. Figur 2A er et fly...

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 proteomikk metode ved hjelp av teknikken av stabile isotoper merking av aminosyrer i cellekultur (SILAC) for å analysere virkningene av HIV-1-infeksjon på verten exosomal proteom. Denne protokollen kan enkelt tilpasses til cellene under ulike stress eller smitteforhold.

SILAC basert proteomikk Karakterisering av Exosomes fra HIV-1 infiserte 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 av HIV-1 Cores

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 verktøy for å utvide Regulatoriske T-celler fra HIV-1-infiserte 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.

Vurdere Medfødt Sensing av HIV-1 infiserte CD4<sup&gt; +</sup&gt; T-celler ved Plasmacytoid dendrittiske celler ved hjelp av en<em&gt; Ex vivo</em&gt; Co-kultursystem.

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 Evaluering av HIV-1 Trimeric kappeglykoproteiner Ved hjelp av en Cell-basert ELISA-analysen

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 fra Plasma av 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. ...