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

이 작품은 BR의 수명 / 술 / 브라운 CFAR, P30AI042853-13S1, NIH P20GM103421, P01AA019072, R01HD072693 및 K24HD080539에 ARRA 보충에 의해 지원되었다. 이 작품은 또한 수명 파일럿 연구 기금 (# 701 5857),로드 아일랜드 재단 의학 연구 그랜트 (# 20133969)와 ML에 NIH 코 브레 URI / RIH 파일럿 연구 그랜트 (P20GM104317)에 의해 지원되었다. 우리는 원고와 그림 준비에 도움 제임스 오스트레일리아 산 아카시아와 VY 젠장 감사합니다.

1. 세포 배양 및 HIV-1 감염 참고 : 실험을 시작하기 전에,이 MTT 분석 (12)를 통해 트리 판 블루 염색 (11)를 통해 세포의 생존과 증식을 확인하는 것이 좋습니다. 또한 새롭게 준비 SILAC 매체를 사용하는 것이 중요합니다. 다양한 세포주가 활발하게 증식 단계에 있으며, HIV-1 감염, 또는 원하는 시험 조건에 민감한만큼 사용될 수있다. 이 프로토콜?...

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이 논문에 기재된 절차에서는 호스트 exosomal 프로테옴에서 HIV-1 감염의 효과를 조사하기 SILAC 기술의 적용을 설명했다. 처음에 감염되지 않은 및 HIV-1에 감염된 세포는 차동 동위 원소 표지입니다. 차등 표지 엑소 좀은 단백질 추출을 수행하기 전에 정제된다. 다음에, 액체 크로마토 그래피 - 탠덤 질량 분석은 exosomal 프로테옴을 분석하기 위해 사용된다. 마지막으로, 생성 된 질량 분석 데이터 및 잠...

바이러스 감염을 포함한 많은 인간의 질병은 종종 및 영향을받는 세포 주위에 발생하는 독특한 세포 과정과 연결되어 있습니다. 단백질은 종종 이러한 과정을 중재 궁극적 이펙터 세포로서 작용. 단백질의 분석은 종종 영향을 세포의 지역 환경에 관한 귀중한 정보를 제공하고 질병 발병의 기본 메커니즘을 이해하는 데 도움이 있습니다. 다양한 단백질 분석 기법 중 프로테오믹스 특히 큰 약속을 보유하고있다. 강력한 대규모 도구로서 특히 프로테오믹스 기능과 단백질의 상호 작용의 영역에서 세포 과정의 체계적인 이해를 제공 할 수있다. 특정 단백질을 분석하여 조사 부위에서 연구자들은 세포 성분, 특히 단백질의 발현을 모니터링 할 수 있도록 분류 기술의 개발을 간단하게한다. 많은 프로테옴 분석은 세포에서 수행되었지만프로테옴 규모, 세포 내 구획에 단백체 특성화 1 특히 유익한 것으로 판명했다. 이는 HIV-1 감염의 연구에서 잘들 수있다. 엑소 좀 셀 타입 2, 3, 광범위한 분비 30-100 nm의 막 소포 간 통신 및 분자 수송의 중요한 구성 요소이다. 이들은 이전에 HIV-1 신진 공정 4, 5에 중요한 역할을하는 발견되었다. 기능 해부와 단백체 분석을 결합함으로써, 우리는 HIV-1에 감염된 세포에서 방출 엑소 좀은 세포 사멸 및 증식 (6)을 포함하여 수용 세포를 이웃에 휴대 속성에 영향을 미칠 독특하고 정량적으로 다른 단백질 서명과 항구 규제 분자로 구성되어 있음을 발견했다. 방법이 프로토콜에 설명되어즉 SILAC HIV-1에 감염된 세포에서 엑소 좀 7 기반 단백체 특성 (세포 배양에서 아미노산에 의해 안정 동위 원소 라벨). 비슷한 접근 방식은 더 나은 특정 구획 또는이자의 일부에 실험 스트레스를 조절하고 설명 된 절차에 필요한 사항을 변경하여 발병하는 동안 다른 세포 내 구획을 이해하고 적용 할 수 있습니다. 정량 프로테오믹스 방법의 최근 발전을 감안할 때, 특정 실험을 위해 가장 효율적인 방법을 선택 할 때 선택할 많은있다. 이 중 화학 기반 iTRAQ (상대 및 절대 정량 동중 태그) (8) 및 라벨없는 MRM (다중 반응 모니터링) (9) 기술이다. 두 방법 모두 강력한 도구이며 특정 설정을위한 좋은 선택입니다. 일반적인 실험실은 주로 세포 라인 작업을 위해, 그러나,이 두 가지 방법이 relativel이Y 높은 비용은 더 많은 시간이 걸리는 SILAC 기반 방법에 비해. SILAC 세포질 단백질로 배양 배지로부터 아미노산의 비 방사성 동위 형태를 포함 대사 라벨링 기반 기술이다. 일반적으로 SILAC 실험 예를 들어 두 개의 세포 집단, 감염 및 감염되지 않은 시작합니다. 전체 라벨이 달성 될 때까지 각각 차등의 특정 동위 원소 환경에서 레이블이 붙어 있습니다. 이러한 세포의 표지 엑소 좀이어서 단백질 추출을 실시한다. 일단 분류 exosomal 단백질 액체 크로마토 그래피 탠덤 질량 분광법 (10)를 사용하여 분석된다 추출 하였다. 마지막으로, 질량 분석 결과와 유의 표지 단백질은 통계를 실시하고 정보학 엄격한 생화학 검증뿐만 아니라 분석한다. 이전 조사 보고서 세포주는 일반적되는 것과 SILAC / 엑소 절차, 일차 전지보다 세포주에 더 적합한 것으로 제안효율적인 동위 원소 라벨에 대한 활성 증식 상태,

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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>
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			<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.

도 1a는 SILAC 라벨링 방법 (21)을 개략적으로 나타내는 흐름도이다. 엑소 좀을 정제하기 위해, 샘플은 원심 분리를 통해 스핀 다운되어야한다. 도 1b는 직렬 초 원심 분리하여 엑소 좀 21 정화 단계를 도시한다. 정제하면 절차에 설명 된 바와 같이, 엑소 좀 실험 단백체 분석 될 수 있습니다. <p class="jove_content" fo:k...

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

여기서는 호스트 exosomal의 프로테옴에서 HIV-1 감염의 효과를 분석하기 위해, 세포 배양 (SILAC)의 아미노산이 안정 동위 원소에 의한 표지의 기술을 이용하여 정량적 프로테오믹스 방법을 설명한다. 이 프로토콜은 쉽게 다른 스트레스 또는 감염 조건 하에서 세포에 적용 할 수있다.

HIV-1에 감염된 세포에서 엑소 좀의 SILAC 기반 단백체 특성

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

그린 형광 단백질을 표현 레브 종속 Lentiviral 벡터를 사용하여 HIV - 1 양성 세포의 특정 마킹

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, ...

연구

면역학 및 감염병학

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

V3의 HIV - 1 Tropism의 Genotypic 추론 사용 인구를 기반 시퀀싱

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

준비 및 사용 HIV - 1 차 감염된 CD4 + 대상 세포로 T 세포 자연 킬러 세포 세포 독성 Assays에서

 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

표준 임상 Assays에 의해 탐지의 한도 이하 바이러스성로드와 HIV 감염된 개인의 확장 및 HIV - 1 RNA를 Quantifying

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

HIV - 1 코어의 시험 관내 Uncoating에서

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

새로운 도구는 HIV-1에 감염된 개인으로부터 규제 T 세포를 확장하려면

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.

HIV-1 감염 CD4의 타고난 감지 평가<sup&gt; +</sup&gt;를 사용하여 형질 수지상 세포에 의한 T 세포<em&gt; 예 생체 내</em&gt; 공동 문화 시스템.

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

셀 기반의 ELISA 분석을 사용하여 HIV-1 삼량 봉투 당 단백질의 구조적 평가

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

HIV-1 양성 개인의 플라즈마에서 엑소 좀의 분리

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. ...