Name: 심혈관 바이오마커 발견을 위한 혈장 유래 세포외 소포체의 분리, 특성화 및 단백질체 분석 (동영상) | JoVE Journal
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Description: 학습하세요 인간 혈장 유래 세포외 소포체(EV)의 분리 및 특성화 방법을 통해 심혈관 바이오마커를 발견하는 기술을. LC-MS/MS 단백질체 분석으로 임상 응급실 환경에서 재현 가능한 결과를 얻을 수 있습니다.

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<meta charset="utf8"></head><body>Plasma Extracellular Vesicle Proteome을 사용한 심혈관 바이오마커 연구

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<li>Nieuwland, R., Siljander, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+beginner%27s+guide+to+study+extracellular+vesicles+in+human+blood+plasma+and+serum%5BTitle%5D)+AND+%22J+Extracell+Vesicles%22%5BJournal%5D)">A beginner's guide to study extracellular vesicles in human blood plasma and serum.</a> J Extracell Vesicles. 13 (1), e12400(2024).</li>
<li>Lacroix, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Impact+of+pre-analytical+parameters+on+the+measurement+of+circulating+microparticles%3A+Towards+standardization+of+protocol%5BTitle%5D)+AND+%22J+Thromb+Haemost%22%5BJournal%5D)">Impact of pre-analytical parameters on the measurement of circulating microparticles: Towards standardization of protocol.</a> J Thromb Haemost. 10 (3), 437-446 (2012).</li>
<li>Coumans, F. aW., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Methodological+guidelines+to+study+extracellular+vesicles%5BTitle%5D)+AND+%22Circ+Res%22%5BJournal%5D)">Methodological guidelines to study extracellular vesicles.</a> Circ Res. 120 (10), 1632-1648 (2017).</li>
<li>Clayton, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Considerations+towards+a+roadmap+for+collection%2C+handling+and+storage+of+blood+extracellular+vesicles%5BTitle%5D)+AND+%22J+Extracell+Vesicles%22%5BJournal%5D)">Considerations towards a roadmap for collection, handling and storage of blood extracellular vesicles.</a> J Extracell Vesicles. 8 (1), 1647027(2019).</li>
<li>Lucien, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Miblood-EV%3A+Minimal+information+to+enhance+the+quality+and+reproducibility+of+blood+extracellular+vesicle+research%5BTitle%5D)+AND+%22J+Extracell+Vesicles%22%5BJournal%5D)">Miblood-EV: Minimal information to enhance the quality and reproducibility of blood extracellular vesicle research.</a> J Extracell Vesicles. 12 (12), e12385(2023).</li>
<li>Zhang, X., Takeuchi, T., Takeda, A., Mochizuki, H., Nagai, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparison+of+serum+and+plasma+as+a+source+of+blood+extracellular+vesicles%3A+Increased+levels+of+platelet-derived+particles+in+serum+extracellular+vesicle+fractions+alter+content+profiles+from+plasma+extracellular+vesicle+fractions%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Comparison of serum and plasma as a source of blood extracellular vesicles: Increased levels of platelet-derived particles in serum extracellular vesicle fractions alter content profiles from plasma extracellular vesicle fractions.</a> PLoS One. 17 (6), e0270634(2022).</li>
<li>Gelibter, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+impact+of+storage+on+extracellular+vesicles%3A+A+systematic+study%5BTitle%5D)+AND+%22J+Extracell+Vesicles%22%5BJournal%5D)">The impact of storage on extracellular vesicles: A systematic study.</a> J Extracell Vesicles. 11 (2), e12162(2022).</li>
<li>Sluijter, J. P. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Extracellular+vesicles+in+diagnostics+and+therapy+of+the+ischaemic+heart%3A+Position+paper+from+the+working+group+on+cellular+biology+of+the+heart+of+the+European+Society+of+Cardiology%5BTitle%5D)+AND+%22Cardiovasc+Res%22%5BJournal%5D)">Extracellular vesicles in diagnostics and therapy of the ischaemic heart: Position paper from the working group on cellular biology of the heart of the European Society of Cardiology.</a> Cardiovasc Res. 114 (1), 19-34 (2018).</li>
<li>Davidson, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Methods+for+the+identification+and+characterization+of+extracellular+vesicles+in+cardiovascular+studies%3A+From+exosomes+to+microvesicles%5BTitle%5D)+AND+%22Cardiovasc+Res%22%5BJournal%5D)">Methods for the identification and characterization of extracellular vesicles in cardiovascular studies: From exosomes to microvesicles.</a> Cardiovasc Res. 119 (1), 45-63 (2023).</li>
<li>Karimi, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Detailed+analysis+of+the+plasma+extracellular+vesicle+proteome+after+separation+from+lipoproteins%5BTitle%5D)+AND+%22Cell+Mol+Life+Sci%22%5BJournal%5D)">Detailed analysis of the plasma extracellular vesicle proteome after separation from lipoproteins.</a> Cell Mol Life Sci. 75 (15), 2873-2886 (2018).</li>
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<li>Gemoll, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Protein+profiling+of+serum+extracellular+vesicles+reveals+qualitative+and+quantitative+differences+after+differential+ultracentrifugation+and+ExoQuick+TM+isolation%5BTitle%5D)+AND+%22J+Clin+Med%22%5BJournal%5D)">Protein profiling of serum extracellular vesicles reveals qualitative and quantitative differences after differential ultracentrifugation and ExoQuick TM isolation.</a> J Clin Med. 9 (5), 1429(2020).</li>
<li>Kreimer, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mass-spectrometry-based+molecular+characterization+of+extracellular+vesicles%3A+Lipidomics+and+proteomics%5BTitle%5D)+AND+%22J+Proteome+Res%22%5BJournal%5D)">Mass-spectrometry-based molecular characterization of extracellular vesicles: Lipidomics and proteomics.</a> J Proteome Res. 14 (6), 2367-2384 (2015).</li>
<li>Shevchenko, A., Tomas, H., Havlis, J., Olsen, J. V., Mann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In-gel+digestion+for+mass+spectrometric+characterization+of+proteins+and+proteomes%5BTitle%5D)+AND+%22Nat+Protoc%22%5BJournal%5D)">In-gel digestion for mass spectrometric characterization of proteins and proteomes.</a> Nat Protoc. 1 (6), 2856-2860 (2006).</li>
<li>Bateman, N. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Maximizing+peptide+identification+events+in+proteomic+workflows+using+data-dependent+acquisition+%28dda%29%5BTitle%5D)+AND+%22Mol+Cell+Proteomics%22%5BJournal%5D)">Maximizing peptide identification events in proteomic workflows using data-dependent acquisition (dda).</a> Mol Cell Proteomics. 13 (1), 329-338 (2014).</li>
<li>Pino, L. K., Just, S. C., Maccoss, M. J., Searle, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Acquiring+and+analyzing+data+independent+acquisition+proteomics+experiments+without+spectrum+libraries%5BTitle%5D)+AND+%22Mol+Cell+Proteomics%22%5BJournal%5D)">Acquiring and analyzing data independent acquisition proteomics experiments without spectrum libraries.</a> Mol Cell Proteomics. 19 (7), 1088-1103 (2020).</li>
</ol>

<table><tbody><tr><td>4x Laemmli 시료 버퍼</td><td>Bio-rad</td><td>1610747</td><td></td></tr><tr><td>10x RIPA-Buffer</td><td>Abcam</td><td>ab156034</td><td></td></tr><tr><td>26.3 mL 폴리카보네이트 병(초원심분리용 캡 어셈블리 포함</td><td>) Beckman Coulter</td><td>355654</td><td></td></tr><tr><td>5810R 벤치탑 원심분리기</td><td>Eppendorf</td><td>5811000015</td><td></td></tr><tr><td>Acetonitrile (ACN)</td><td>Biosolve</td><td>0001204101BS</td><td></td></tr><tr><td>디티오트레이톨(DTT)</td><td>시그마-알드리치</td><td>43816-10ML</td><td></td></tr><tr><td>둘베코의 인산염 완충 식염수(PBS)</td><td>시그마-알드리치</td><td>D8537</td><td></td></tr><tr><td>포름산 99% ULC/MS 100</td><td>Biosolve</td><td>0006914143BS</td><td></td></tr><tr><td>히스토파크 - 1077</td><td>시그마-알드리치</td><td>10771</td><td></td></tr><tr><td>요오도아세트아미드(IAA)</td><td>시그마-알드리치</td><td>I6125-5G</td><td></td></tr><tr><td>질량분석기 Orbitrap Q Exactive HF</td><td>Thermo Fisher Scientific</td><td>IQLAAEGAAPFALGMBDK</td><td></td></tr><tr><td>MOPS SDS 러닝 버퍼</td><td>Thermo Fisher</td><td>B0001</td><td></td></tr><tr><td>NanoSight NS300</td><td>Malvern Panalytical&nbsp;</td><td>NS300</td><td></td></tr><tr><td>Nanosight NTA 3.2 소프트웨어</td><td>Malvern Panalytical&nbsp;</td><td></td><td></td></tr><tr><td>누페이지 4&nbsp; bis 12&nbsp;%, Bis-Tris 단백질 겔</td><td>Invitrogen</td><td>NP0323</td><td></td></tr><tr><td>Optima XPN-80 플로어스탠딩 초원심분리기</td><td>Beckman Coulter</td><td>521-4180</td><td></td></tr><tr><td>PageRuler Plus Prestained Protein Ladder</td><td>Thermo Fisher Scientific</td><td>26619</td><td></td></tr><tr><td>프로테아제/포스파타제 억제제 칵테일 (100X)</td><td>Cell Signaling Technology</td><td>5872S</td><td></td></tr><tr><td>단백질 마커</td><td>Thermo Fisher</td><td>26616</td><td></td></tr><tr><td>Proteom Discoverer 2.5</td><td>Thermo Fisher</td><td></td><td></td></tr><tr><td>Q Exactive Plus 하이브리드 사중극자-Orbitrap 질량분석기</td><td>Thermo Fisher Scientific</td><td>IQLAAEGAAPFALGMBDK</td><td></td></tr><tr><td>빠른 염색 coomasssie</td><td>Serva</td><td>35081.01</td><td></td></tr><tr><td>ReproSil-Pur 120 C18-AQ, 1.9 &마이크로; m 1 g</td><td>Dr. Maisch</td><td>r119.aq.0001</td><td></td></tr><tr><td>SDS-PAGE 상업용 젤</td><td>Thermo Fisher</td><td>NW00100BOX</td><td></td></tr><tr><td>S-Monovette&nbsp; Citrat 9NC 0.106 mol/l 3,2%</td><td>Sarstedt</td><td>02.1067.001</td><td></td></tr><tr><td>Speed vac concentrator</td><td>Savant</td><td></td><td></td></tr><tr><td>Swiss-Prot (Uniprot) Homo sapiens (UP000005640, 2020년 6월) 단백질 데이터베이스</td><td>UniProt</td><td>https://www.uniprot.org/proteomes/UP000005640</td><td></td></tr><tr><td>Triethylammonium bicarbonate buffer (TEAB)</td><td>Sigma-Aldrich</td><td>T7408</td><td></td></tr><tr><td>Trifluoroacetic acid (TFA) HPLC >99%, 100mL</td><td>Sigma-Aldrich</td><td>302031-100ML</td><td></td></tr><tr><td>Trypsin MS 등급</td><td>Thermo Fisher</td><td>90058</td><td></td></tr><tr><td>유형 50.2 Ti 고정각 로터</td><td>Beckman Coulter</td><td>337901</td><td></td></tr><tr><td>UHPLC Dionex Ultimate 3000</td><td>Thermo Fisher Scientific</td><td>ULTIM3000RSLCNANO</td><td></td></tr><tr><td>Water</td><td>Biosolve</td><td>0023214102BS</td><td></td></tr></tbody></table>

심혈관 바이오마커 발견을 위한 혈장 유래 세포외 소포체의 분리, 특성화 및 단백질체 분석

Isolation, Characterization, and Proteomic Analysis of Plasma-Derived Extracellular Vesicles for Cardiovascular Biomarker Discovery

EV는 확립된 차등 초원심분리 프로토콜을 사용하여 명백한 심혈관 질환이 없는 환자의 혈장 샘플(n = 3)과 비ST 상승 심근 경색(NSTEMI; n = 2) 환자로부터 분리되었습니다. 혈장 EV의 적절한 분리는 동일한 환자의 EV 고갈 혈장과 비교하여 EV 분리물에서 EV 농축 단백질 TSG-101, Annexin 5(Anx5) 및 CD9의 면역 블로팅에 의해 확인되었습니다(그림 1A). 음성 대조군 역할을 하는 트랜스페린은 EV가 고갈된 혈청 샘플에서 발견되었지만 EV 분리물에서는 검출되지 않아 구획의 적절한 분리를 확인했습니다. 분리된 혈장 샘플에서 일반적인 혈소판 마커인 인테그린 β3와 지단백질 오염 마커인 아포지단백 A1(ApoA1)에 대한 면역 블로팅은 혈액 처리 후 심각한 오염을 나타내지 않았습니다(보충 그림 1).
나노입자 추적 분석을 사용하여 브라운 운동을 기반으로 EV를 식별하고...

Extracellular vesicles (EV) are cell-derived, lipid bilayer-enclosed, non-replicable nanoparticles. EV currently gain attention in cardiovascular research ...

isolation-characterization-proteomic-analysis-plasma-derived

Research

연구

JoVE Journal

Medicine

의학

혈액에 존재하는 세포외 소포체는 다양한 질병에 대한 새로운 바이오마커로 제안되었습니다. 여기에서는 말초 혈액 샘플에서 대형 원형질막 유래 미세소포를 분리하고 기존 유세포 분석 및 웨스턴 블로팅(Western Blotting)을 통한 후속 분석을 위한 프로토콜을 제시합니다.

말초 혈액에서 마이크로 소포의 분리 및 특성

여기, 선물이 exosomes 플라즈마와 비 exosomal 혈액 단백질의 감소 공동 정화와 혈 청에서 순화 하는 프로토콜. 최적화 된 프로토콜 적용, 효소 치료, 및 크기 배타 착 색 인쇄기를 포함 한다. Exosomes 혜택 다운스트림 분석, 소포 및 proteomic 특성의 더 정확한 정량화를 포함 하 여 향상 된 정화.

단백질 소화, 한, 및 크기 배제 크로마토그래피 인간의 혈장 및 혈 청에서 Exosomes의 절연을 최적화 하

프로토콜 셀 문화 supernatants 함께 사용 하기 위해 작은 extracellular 소포 (EV)에 표면 epitopes를 감지 하도록 설계 된 재현 방법을 설명 합니다. 그것은 항 체를 표면 항 원 CD9, 인식 CD81 그리고 CD63, 함께 하는 비즈를 사용 하 여 특정 EV immunoprecipitation 활용. 메서드는 다운스트림 흐름 cytometry 분석에 최적화 되어 있습니다.

Extracellular 소포 세포 조절 미디어에서의 Cytometric 분석 흐름

본 보고서는 마이크로글리아 또는 혈액 대식세포로부터 세포외 소포(EV) 격리에 대한 연대순 요구 사항을 강조합니다. Microglia-파생된 EV는 혈액 대식세포 유래 EV가 시험관내 분석에서 C6 신경교종 세포 침입의 대조군에서 연구된 동안 신경이대 발생의 조절자로 평가되었다. 목표는 특정 미세 환경에서 면역 중재자로서 이러한 EV 기능을 더 잘 이해하는 것입니다.

면역 세포 유래 세포 외 소포의 특성화 및 세포 환경에 대한 기능적 영향 연구

본 프로토콜은 표면 항원 및 단백질 화물의 후속 프로파일링과 함께 말초 혈액 및 고형 조직으로부터 세포외 소포를 추출하는 방법을 설명한다.

혈장 및 고형 조직에서 추적 가능하고 기능화된 세포외 소포의 분리 및 분석

최신 세대의 EV 특성화 도구는 여러 파라미터에 대한 단일 EV 분석이 동시에 가능합니다. 나노유세포분석은 표지 없이 45nm보다 큰 모든 생물학적 입자를 측정하고 다양한 형광 표지 기술로 아집단의 특정 특성을 식별합니다.

나노유세포분석을 통한 단일 세포외 소포 막횡단 단백질 특성 분석

본 프로토콜은 기능성 자성 비드를 이용한 요로 세포외 소포체의 효율적인 분리를 위한 상세한 설명을 제공한다. 또한 웨스턴 블로팅(western blotting), 단백질체학(proteomics) 및 포스포프로테오믹스(phosphoproteomics)를 포함한 후속 분석도 포함됩니다.

단백질체학 및 인산염체학 분석을 위한 생체 유체에서 세포외 소포체의 화학적 친화성 기반 분리

단백질체학 실험에 적합한 세포외 소포체 분리를 수행하기 위해 폴리카보네이트 초원심분리기 튜브를 세척하고 재사용하기 위한 자세한 프로토콜이 제공됩니다.

폴리카보네이트 초원심분리기 튜브는 세포외 소포체의 단백질체 분석에 재사용됩니다.

여기에서는 인간 피험자의 눈물 및 타액과 같은 생물학적 액체에서 수집된 세포외 소포체(EV)의 특성화 방법을 보여줍니다. 이 방법에 사용된 스캐너는 샘플의 1μL에서 EV의 표현형, 크기 및 총 입자 수를 감지할 수 있습니다.

Characterizing Extracellular Vesicles from Biological Fluids(생물학적 유체로부터 세포외 소포체 특성화)

이 동영상은 사후의 눈에 인간의 유리 신체의 다양한 반투명 구조를 차별하고 해부를위한 효과적인 기법을 보여줍니다.

Proteomic 분석을위한 인적 유리 바디 요소의 해부

학습하세요 인간 혈장 유래 세포외 소포체(EV)의 분리 및 특성화 방법을 통해 심혈관 바이오마커를 발견하는 기술을. LC-MS/MS 단백질체 분석으로 임상 응급실 환경에서 재현 가능한 결과를 얻을 수 있습니다.

심혈관 바이오마커 발견을 위한 혈장 유래 세포외 소포체의 분리, 특성화 및 단백질체 분석

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