이 연구는 사이먼 프레이저 대학의 시작 기금에 의해 지원되었다.

1. 수확 막 <ol><li> 세포 펠렛 (~ 10 8 셀)에 프로테아제 억제제 칵테일 저장성 버퍼 (10 MM 트리스 - 염산, 10 밀리미터의 KCl, 1.5 mM의 MgCl2를, 산도 8.0)을 추가하고 얼음에 15 ~ 30 분 동안 품어. 를 Lyse 주사기 바늘 (5 ~ 10 패스)를 통해 샘플을 통과하거나 다운스 호모 게 나이저 (15 ~ 30 타)로 샘플을 균질화하여 세포. 용해의 효율을 확인하는 혈구와 트리 판 블루 염색법을 사용합니다. </li><li> 3000 GX 15 분에 용해 액의 차등 원심 분리하여 미세 소체 분획을 확보 한 다음에 10 GX 2 시간. 우선 원심 분리 핵 분획 인 펠렛을 취득하고, 상층 액의 원심 분리 이후의 원형질막뿐만 아니라 막 - 결합 세포 기관 (8)를 포함 마이크로 솜 분획 인 제 펠릿을 생성한다. 최종 상층 액을 세포질 부분입니다. 더 분​​석가를위한 -80 ° C의 냉동고에 미세 소체 분수를 저장아래와 같습니다. </li></ol> 2., 용해 변성, 소화 막 단백질 <ol><li> 변성 버퍼 (40 MM 트리스, 10 mM의 EDTA, 10 mM의 TCEP, 0.5 % Rapigest, 산도 8)에서 마이크로 솜 분획을 용해 후 10 분 동안 100 ° C에서 솔루션을 품어. </li><li> 실온으로 가열 된 용액을 냉각 에이트 M의 최종 농도로 용액에 초 고순도 우레아 분말을 추가하고 30 분 동안 37 ° C에서 샘플을 배양한다. </li><li> 15 mM의 최종 농도에 샘플에 요오도 아세트 아미드 스톡 용액을 추가하고, 시료에 자유 티올을 알킬화 실온에서 30 분 동안 어두운 용액을 배양한다. 10 mM의 최종 농도 시료에 DTT 주식 솔루션을 추가하고 과도한 요오도 아세트 아미드를 해소하기 위해 실온에서 또 다른 10 분 동안 솔루션을 품어. </li><li> pH를 8에서 40 mM 트리스 완충액으로 얻어진 용액을 10 배 희석하고,이어서 trypsi 1:20의 비율로 샘플을 트립신을 쓰총 단백질 명. 반응이 완료 될 수 있도록 37 ° C 오븐에서 밤새에서 소화 반응을 유지한다. </li><li> 즉 Rapigest 염산, 또한 세제를 분해 조건으로 pH 1로 시료 용액을 산성화하여 소화를 종료합니다. 1 시간 동안 37 ° C에서 Rapigest을 저하하고, 원심 분리에 의해 개발 된 침전을 제거합니다. </li><li> C-18 고체상 추출 (SPE) 카트리지에 의해 샘플 펩티드를 포함하고를 Speed​​vac에 의해 얻어진 샘플을 건조 뜨는을 청소합니다. </li><li> 이전과 소화 효율을 확인하는 트립신 분해 된 후 샘플의 SDS-PAGE 분석을 수행합니다. </li></ol> 3. 글리코 펩티드 캡처 <ol><li> 결합 완충액 (100 mM의 아세트산 나트륨, pH를 5.5)에서 세정 펩티드를 용해. 실온에서 어두운 곳에서 30 분 동안 10 mM의 최종 농도에서 펩티드 용액에과 요오드 산 나트륨을 추가. 이 알데히드를 글리 칸의 시스 - 디올기를 산화 할 것이다,하는 하이 드라 지드 화학을 통해 수지 커플 글리 칸이 있습니다. 실온에서 10 분 동안 20 mM의 최종 농도 및 pH를 5로 황산나트륨으로 과도한 요오 담금질. </li><li> 수지에 커플 글리코 펩타이드 1:4 (용액에 수지)의 비율 펩티드 용액에 히드라 지드 - 유도 된 수지를 소개한다. 완전한 연결을위한 엔드 - 투 - 엔드 회전 1-2 일 동안 37 ° C에서 반응을 품어. </li><li> 다음의 각 1 ㎖로 두 번 수지를 세척하여 언 바운드 펩티드를 제거 DI 물, 1.5 M 염화나트륨, 메탄올 80 % 아세토 니트릴. 마지막으로, 100 ㎜ NH 4 HCO 3 시스템의 완충액을 교환하고 pH를 8에서 100 ㎜ NH 4 HCO 3 1 ㎖와 수지 3 회 세척 하였다. <ol><li> (옵션) 언 바운드 펩티드의 분석을위한 뜨는 세척을 수집합니다. </li></ol></li><li> 와 37 ° C에서 하룻밤 배양에 인, PNGase F로 수지에서 N-글리코 펩타이드를 해제N 엔드 - 투 - 엔드 회전. 원심 분리 및 80 % 아세토 니트릴 세척에 의해 방출 된 펩티드를 수집합니다. LC-MS 분석을 위해를 Speed​​vac에서 얻어진 용액을 건조시켰다. </li></ol> 4. 또한 분별 (선택 사항) 상기 샘플의 복잡성을 단순화하기 위해, 얻어진 N-글리코 펩티드 분획. 예를 들어, 10 mM의 암모니아 메이트, 20 % 아세토 니트릴을 사용하여 pH 3으로 건조 펩티드를 재용 및 펩티드 분획 강한 양이온 교환 (SCX)을 크로마토 그래피를 사용한다. 얻어진 용출액을 건조하고 역상 LC-MS 8,17,18에 의해 얻어진 펩티드 분수를 분석 할 수 있습니다. 출시 N-글리코 펩티드 5. 청소 (옵션) 문제는 펩티드의 잠재적 인 오염 상승하면 0.1 % 포름산으로 건조 된 펩티드를 재용하고 추가 할 수 역상 LC-MS 분석 이전에 펩티드를 청소 MCX SPE 열을 사용합니다. <stronG&gt; 참고 : 데이터베이스 검색 매개 변수를. 수지 오프 N-글리코 펩타이드를 선택적으로 절단하는 동안, 인, PNGase F는 아스파르트 산에 N-글리 칸 연결 아스파라긴을 변환합니다. 따라서 해방 된 N-글리코 펩타이드의 0.9840 다 질량 변화가있다. 정확하게이 펩티드를 확인하려면,이 수정은 메티오닌, 시스테인 산화의 carbamidomethylation 일반적인 수정과 함께 검색 매개 변수를 추가해야합니다.

<ol>
	<li>Cho, W., Stahelin, R. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane-protein+interactions+in+cell+signaling+and+membrane+trafficking.">Membrane-protein interactions in cell signaling and membrane trafficking.</a> Annual review of biophysics and biomolecular structure. 34, 119-151 (2005).</li><li>Tadevosyan, A., Vaniotis, G., Allen, B. G., Hebert, T. E., Nattel, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=protein-coupled+receptor+signalling+in+the+cardiac+nuclear+membrane:+evidence+and+possible+roles+in+physiological+and+pathophysiological+function.">protein-coupled receptor signalling in the cardiac nuclear membrane: evidence and possible roles in physiological and pathophysiological function.</a> The Journal of physiology. 590, 1313-1330 (2012).</li><li>White, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biophysical+dissection+of+membrane+proteins.">Biophysical dissection of membrane proteins.</a> Nature. 459, 344-346 (2009).</li><li>Heijne, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane-protein+topology.+.">Membrane-protein topology. .</a> Nature reviews Molecular cell biology. 7, 909-918 (2006).</li><li>Cox, J., Mann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+high-resolution+proteomics+for+data-driven+systems+biology.">Quantitative high-resolution proteomics for data-driven systems biology.</a> Annual review of biochemistry. 80, 273-299 (2011).</li><li>Lamond, A. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advancing+cell+biology+through+proteomics+in+space+and+time+(PROSPECTS).">Advancing cell biology through proteomics in space and time (PROSPECTS).</a> Mol Cell Proteomics. 11, (2012).</li><li>Savas, J. N., Stein, B. D., Wu, C. C., Yates 3rd, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass+spectrometry+accelerates+membrane+protein+analysis.">Mass spectrometry accelerates membrane protein analysis.</a> Trends in biochemical sciences. 36, 388-396 (2011).</li><li>Sun, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shotgun+glycopeptide+capture+approach+coupled+with+mass+spectrometry+for+comprehensive+glycoproteomics.">Shotgun glycopeptide capture approach coupled with mass spectrometry for comprehensive glycoproteomics.</a> Mol Cell Proteomics. 6, 141-149 (2007).</li><li>Lowe, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycosylation,+immunity,+and+autoimmunity.">Glycosylation, immunity, and autoimmunity.</a> Cell. 104, 809-812 (2001).</li><li>Dodds, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gas-phase+dissociation+of+glycosylated+peptide+ions.">Gas-phase dissociation of glycosylated peptide ions.</a> Mass spectrometry reviews. 31, 666-682 (2012).</li><li>Kaji, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lectin+affinity+capture,+isotope-coded+tagging+and+mass+spectrometry+to+identify+N-linked+glycoproteins.">Lectin affinity capture, isotope-coded tagging and mass spectrometry to identify N-linked glycoproteins.</a> Nat Biotechnol. 21, 667-672 (2003).</li><li>Zhang, H., Li, X. J., Martin, D. B., Aebersold, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+quantification+of+N-linked+glycoproteins+using+hydrazide+chemistry,+stable+isotope+labeling+and+mass+spectrometry.">Identification and quantification of N-linked glycoproteins using hydrazide chemistry, stable isotope labeling and mass spectrometry.</a> Nat Biotechnol. 21, 660-666 (2003).</li><li>Hagglund, P., Bunkenborg, J., Elortza, F., Jensen, O. N., Roepstorff, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+strategy+for+identification+of+N-glycosylated+proteins+and+unambiguous+assignment+of+their+glycosylation+sites+using+HILIC+enrichment+and+partial+deglycosylation.">A new strategy for identification of N-glycosylated proteins and unambiguous assignment of their glycosylation sites using HILIC enrichment and partial deglycosylation.</a> J Proteome Res. 3, 556-566 (2004).</li><li>Bond, M. R., Kohler, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+methods+for+glycoprotein+discovery.">Chemical methods for glycoprotein discovery.</a> Current opinion in chemical biology. 11, 52-58 (2007).</li><li>Pasini, E. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In-depth+analysis+of+the+membrane+and+cytosolic+proteome+of+red+blood+cells.">In-depth analysis of the membrane and cytosolic proteome of red blood cells.</a> Blood. 108, 791-801 (2006).</li><li>Wollscheid, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass-spectrometric+identification+and+relative+quantification+of+N-linked+cell+surface+glycoproteins.">Mass-spectrometric identification and relative quantification of N-linked cell surface glycoproteins.</a> Nat Biotechnol. 27, 378-386 (2009).</li><li>Sun, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=N-Glycoproteome+of+E14.Tg2a+mouse+embryonic+stem+cells.">N-Glycoproteome of E14.Tg2a mouse embryonic stem cells.</a> PLoS ONE. 8, (2013).</li><li>Sun, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycocapture-assisted+global+quantitative+proteomics+(gagQP)+reveals+multiorgan+responses+in+serum+toxicoproteome.">Glycocapture-assisted global quantitative proteomics (gagQP) reveals multiorgan responses in serum toxicoproteome.</a> J Proteome Res. 12, 2034-2044 (2013).</li></ol>

저자는 더 경쟁 재정적 이익이 없다는 것을 선언합니다.

여기에서 우리는 세포 표면 단백질 프로파일에 대한 글리코 펩타이드 캡처 전략을 소개합니다. 방법은 분비 된 혈액에서와 단백질뿐 아니라 기타 체액 또는 세포 배양 배지에서 연구에 적용될 수있다. 방법의 성공은 샘플의 완전 분해에 의존한다; 따라서 소화 효율의 SDS-PAGE 특성화 특히 시료의 처음 분석을 위해 필요하다. 완전한 소화는 막 단백질에 대한 도전 할 수 있고, ?...

세포 표면 단백질은 세포 외 환경과 상호 작용하는 세포의 외부에서 내부로의 신호를 중계. 따라서, 세포 외 기질 단백질을 포함하여 이러한 단백질은 세포 생물학 및 증식, 성장, 이동, 분화에서 등 노화에 이르기까지 생리학의 모든 측면에서 중요한 역할을한다. 표면 단백질이 다른 세포, 단백질과 작은 분자 1-3와 상호 작용에 의해 작동합니다. 약물의 60 % 이상이 세포 표면 단백질 4를 대상으로하는 등 세포 표면 단백질의 분자 특성은 생물 학자뿐만 아니라, 제약 회사를위한뿐만 아니라 큰 관심입니다. 뛰어난 감도, 정확성 및 단백질과 펩티드의 식별을위한 처리량 텐덤 질량 분석법 (MS)는 글로벌 프로테오믹스 연구 5,6를위한 강력한 도구이다. 그러나, 표면 단백질은, MS-기반 프로테오믹스에 상당한 도전을 제기대부분의 표면 단백질은 낮은 수량과 무거운 수정으로 존재한다. 표면 단백질의 막에 걸친 영역들을 소수성 렌더링; 이것은 특히 다중 경로 막 횡단 단백질에 대한 경우이다. 이 세제의 도움없이 수용액에 막 단백질을 용해하는 것이 곤란하다; 그러나 세제의 사용은 일반적으로 단백질 식별의 HPLC와 MS -1,7,8,8의 성능을 억제한다. 따라서, 막 단백질은 가난 직접 LC-MS 기반 프로테오믹스 특징으로하고 있습니다. 포도당은 세포 표면 단백질 9에서 일어나고있는 가장 중요하고 풍부한 번역 후 변형 중 하나입니다. 글리 칸의 엄청난 복잡성과 이질성은 펩티드 &#39;MS 신호 (10)를 방해. 그럼에도 불구하고, 몇 가지 프로테오믹스 방법은 표면의 단백질을 풍부하게하기 전에 LC-MS 분석에 단백질에서 설탕 부분을 제거하는이 독특한 변형을 사용했다. 이 방법의 렉틴 기반 선호도 캡처 (11)과 하이 드라 지드 기반 또는 붕산 기반 화학 캡처 (12)뿐만 아니라 친수성 크로마토 그래피 분리 8,13 있습니다. 글리 칸의 제거는 일반 단백질에 막 단백질을 변환하고 크게 MS의 특성을 단순화합니다. 당화 또한 단백질 막에 대비 높은 용해도를 분비 단백질에서 발생하기 때문에, 많은 glycoproteomic 방법은 수용성 단백질에 최적화 된 낮은 글리코 펩티드 선택과 단백질 8,14를 막에 배포되는 감도를 갖는 경향이있다. 다른 방법도 풍부하게 존재한다, 특히, 초 원심 분리 (15) 및 라벨 (16) 전략을 사용하는 것과 같은 세포 표면 단백질. 막 단백질의 특성에 대한 우리의 방법과 기존의 방법과 자세한 비교는 최근 17를 실시하고, 그 결과는 우리의 방법이 동일하게 수행 할 수있는 표시, 경우에 없습니다모든 비교 막 단백질 체학 방법보다 더 나은,하지만 더 소박하고. 연구팀은이 방법을 사용하기 위해, 여기에 우리 세부 일반 프로토콜입니다. 이 방법은 기존의 glycoproteomics 전략의 몇 가지 장점을 통합하고 막 당 단백질을 위해 특별히 고안되어, 아직 방법은 분비 단백질을 동일하게 작동합니다. 이 방법의 특징은 1) 막 단백질의 완전한 용해, 2) 대신에 고체 포집 기판을 사용하는 경우 잠재적 인 입체 장해를 제거하는 당 단백질의 글리코 펩타이드의 농축, 3) 하이 드라 지드 화학의 사용은 공유 결합 사이를 형성하도록 글리코 펩타이드와 캡처 기판 접합 글리코 펩타이드가 높은 glycoselectivity 엄격한 세척을 견딜 수 있고, 4) 능력이 감소 샘플 손실 및 단축 절차 기간 동안 하나의 튜브 전체 포착 절차를 수행 할 수 있도록. VARI 연구에이 방법을 실행 한 후세포와 조직 등의 생체 시료 중의 ety, 우리는 당 단백질 8,17,18에 높은 선택성 (&gt; 90 %)을 관찰했다.

<table border="0" cellpadding="0" cellspacing="0" width="701">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DTT</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:119px;">646563</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">TCEP</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:119px;">646547</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Iodoacetamide</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:119px;">A3221</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Rapigest SF</td>
			<td style="width:199px;">Waters</td>
			<td style="width:119px;">186001860</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium periodate</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:119px;">311448</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PNGase F</td>
			<td style="width:199px;">New England Biolabs</td>
			<td style="width:119px;">P0704S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Affi-Gel Hz Hydrazide gel</td>
			<td style="width:199px;">Bio-Rad</td>
			<td style="width:119px;">153-6047</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Trypsin</td>
			<td style="width:199px;">Worthington Biomedical</td>
			<td style="width:119px;">LS02115</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sep-Pak C-18 cartridge</td>
			<td style="width:199px;">Waters</td>
			<td style="width:119px;">WAT054955</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Oasis MCX cartridge</td>
			<td style="width:199px;">Waters</td>
			<td style="width:119px;">186000252</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Protease inhibitor coctail</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:119px;">P8340</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Urea</td>
			<td style="width:199px;">Amersco</td>
			<td style="width:119px;">568</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium sulphite</td>
			<td style="width:199px;">Caledon</td>
			<td style="width:119px;">8360-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Invertase</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:119px;">I0408</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Alpha-1 trypsin</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:119px;">F2006</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ribonulease B</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:119px;">R7884</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Avidin</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:119px;">A9275</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ovalbumin</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:119px;">A5503</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Conalbumin</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:119px;">C0755</td>
			<td></td>
		</tr>
	</tbody>
</table>

glycopeptide capture for cell surface proteomics

세포 외 기질 단백질을 포함한 세포 표면 단백질은, 이러한 성장, 분화, 증식 등의 모든 주요 셀룰러 프로세스 및 기능에 참여합니다. 이 단백질의 포괄적 인 특성은 바이오 마커 발견, 세포 유형 식별 및 약물 표적 선택뿐만 아니라, 세포 생물학 및 생리학에 대한 우리의 이해를 사전에 도움 풍부한 정보를 제공합니다. 표면 단백질은, 그러나, 그들의 본질적으로 낮은 풍부, 소수성, 무거운 번역 후 변형의 중요한 분석 과제를 포즈. 표면 단백질에 널리 당화을 활용, 우리는 여기에 몇 가지 기존의 N-glycoproteomics 수단의 장점을 통합하는 높은 처리량 글리코 펩타이드 캡처 방법을 소개합니다. 우리의 방법은 표면 단백질에서 파생 된 글리코 펩타이드를 풍부하게하고 LC-MS를 사용하여 손쉬운 프로테오믹스에 대한 자신의 글리 칸을 제거 할 수 있습니다. 확인 된 N-glycoproteome는 INF를 포함한다단백질의 정체성과 양뿐만 아니라 포도당의 자신의 사이트의 ormation. 이 방법은 암, 줄기 세포, 약물 독성을 포함한 영역에서 일련의 연구에 적용되어왔다. 방법의 한계는 샘플의 상대적으로 많은 양의 세포질 단백질을 중심으로 연구와 비교하여 본 분석에 필요한되도록 표면 막 단백질의 낮은 풍부에있다.

실험 절차의 대표적인 흐름도는도 1에 요약되어있다. 라벨 및 상기 분류 단계는 선택적이며 상세는 최근 간행물 (18)에 기재되어있다. 또 다른 옵션은 수지와 반응하지 않는 비 변형 펩티드를 분석하는 것이다. 변형되지 않은 펩티드를 분석의 장점은 단단한 접합부에서 claudins 같은 비 - 글리코 실화 된 펩티드 및 단백질의 가능성 식별을 포함한다; 추가적인 장점은보다 정확한...

Watch this Scientific Journal Video about Glycopeptide Capture for Cell Surface Proteomics at JoVE.com

Glycopeptide Capture for Cell Surface Proteomics

세포 표면 단백질은 생물학적으로 중요하고 광범위 글리코 실화이다. 우리는 손쉬운 LC-MS 기반 단백체 분석을 위해이 단백질을 용해 풍부하고 deglycosylate 여기 글리코 펩타이드 캡처 방법을 소개합니다.

세포 표면 단백질 체학 (Proteomics)에 대한 글리코 펩타이드 캡처

Cell surface proteins, including extracellular matrix proteins, participate in all major cellular processes and functions, such as growth, ...

glycopeptide-capture-for-cell-surface-proteomics

Research

JoVE Journal

Biology

11.2K 조회수.  Simon Fraser University.  세포 표면 단백질은 생물학적으로 중요하고 광범위 글리코 실화이다. 우리는 손쉬운 LC-MS 기반 단백체 분석을 위해이 단백질을 용해 풍부하고 deglycosylate 여기 글리코 펩타이드 캡처 방법을 소개합니다. 

비디오: Glycopeptide Capture for Cell Surface Proteomics

Cell Capture Using a Microfluidic Device

Microfluidic 장치를 사용하여 셀 캡처

연구

생물학

High-throughput Crystallization of Membrane Proteins Using the Lipidic Bicelle Method

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane bilayer that surrounds all cells and organelles. Alterations in the function of MPs result in many human diseases and disorders; thus, an intricate understanding of their structures remains a critical objective for biological research. However, structure determination of MPs remains a significant challenge often stemming from their hydrophobicity. 
MPs have substantial hydrophobic regions embedded within the bilayer. Detergents are frequently used to solubilize these proteins from the bilayer generating a protein-detergent micelle that can then be manipulated in a similar manner as soluble proteins. Traditionally, crystallization trials proceed using a protein-detergent mixture, but they often resist crystallization or produce crystals of poor quality. These problems arise due to the detergent&prime;s inability to adequately mimic the bilayer resulting in poor stability and heterogeneity. In addition, the detergent shields the hydrophobic surface of the MP reducing the surface area available for crystal contacts. To circumvent these drawbacks MPs can be crystallized in lipidic media, which more closely simulates their endogenous environment, and has recently become a de novo technique for MP crystallization.
Lipidic cubic phase (LCP) is a three-dimensional lipid bilayer penetrated by an interconnected system of aqueous channels1. Although monoolein is the lipid of choice, related lipids such as monopalmitolein and monovaccenin have also been used to make LCP2. MPs are incorporated into the LCP where they diffuse in three dimensions and feed crystal nuclei. A great advantage of the LCP is that the protein remains in a more native environment, but the method has a number of technical disadvantages including high viscosity (requiring specialized apparatuses) and difficulties in crystal visualization and manipulation3,4. Because of these technical difficulties, we utilized another lipidic medium for crystallization-bicelles5,6 (Figure 1). Bicelles are lipid/amphiphile mixtures formed by blending a phosphatidylcholine lipid (DMPC) with an amphiphile (CHAPSO) or a short-chain lipid (DHPC). Within each bicelle disc, the lipid molecules generate a bilayer while the amphiphile molecules line the apolar edges providing beneficial properties of both bilayers and detergents. Importantly, below their transition temperature, protein-bicelle mixtures have a reduced viscosity and are manipulated in a similar manner as detergent-solubilized MPs, making bicelles compatible with crystallization robots. 
Bicelles have been successfully used to crystallize several membrane proteins5,7-11 (Table 1). This growing collection of proteins demonstrates the versatility of bicelles for crystallizing both alpha helical and beta sheet MPs from prokaryotic and eukaryotic sources. Because of these successes and the simplicity of high-throughput implementation, bicelles should be part of every membrane protein crystallographer&prime;s arsenal. In this video, we describe the bicelle methodology and provide a step-by-step protocol for setting up high-throughput crystallization trials of purified MPs using standard robotics.

Bicelles are lipid/amphiphile mixtures that maintain membrane proteins (MPs) within a lipid bilayer but have unique phase behavior that facilitates high-throughput screening by crystallization robots. This technique has successfully produced a number of high-resolution structures from both prokaryotic and eukaryotic sources. This video describes protocols for generating the lipidic bicelle mixture, incorporating MPs into the bicelle mixture, setting up crystallizations trials (manually as well as robotically) and harvesting crystals from the medium.

Lipidic Bicelle 방법을 사용하여 막 단백질의 높은 처리량 결정

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane ...

Avidity-based Extracellular Interaction Screening (AVEXIS) for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions

Extracellular protein:protein interactions between secreted or membrane-tethered proteins are critical for both initiating intercellular communication and ensuring cohesion within multicellular organisms. Proteins predicted to form extracellular interactions are encoded by approximately a quarter of human genes1, but despite their importance and abundance, the majority of these proteins have no documented binding partner. Primarily, this is due to their biochemical intractability: membrane-embedded proteins are difficult to solubilise in their native conformation and contain structurally-important posttranslational modifications. Also, the interaction affinities between receptor proteins are often characterised by extremely low interaction strengths (half-lives &lt; 1 second) precluding their detection with many commonly-used high throughput methods2. 
Here, we describe an assay, AVEXIS (AVidity-based EXtracellular Interaction Screen) that overcomes these technical challenges enabling the detection of very weak protein interactions (t1/2 &le; 0.1 sec) with a low false positive rate3. The assay is usually implemented in a high throughput format to enable the systematic screening of many thousands of interactions in a convenient microtitre plate format (Fig. 1). It relies on the production of soluble recombinant protein libraries that contain the ectodomain fragments of cell surface receptors or secreted proteins within which to screen for interactions; therefore, this approach is suitable for type I, type II, GPI-linked cell surface receptors and secreted proteins but not for multipass membrane proteins such as ion channels or transporters.
The recombinant protein libraries are produced using a convenient and high-level mammalian expression system4, to ensure that important posttranslational modifications such as glycosylation and disulphide bonds are added. Expressed recombinant proteins are secreted into the medium and produced in two forms: a biotinylated bait which can be captured on a streptavidin-coated solid phase suitable for screening, and a pentamerised enzyme-tagged (&beta;-lactamase) prey. The bait and prey proteins are presented to each other in a binary fashion to detect direct interactions between them, similar to a conventional ELISA (Fig. 1). The pentamerisation of the proteins in the prey is achieved through a peptide sequence from the cartilage oligomeric matrix protein (COMP) and increases the local concentration of the ectodomains thereby providing significant avidity gains to enable even very transient interactions to be detected. By normalising the activities of both the bait and prey to predetermined levels prior to screening, we have shown that interactions having monomeric half-lives of 0.1 sec can be detected with low false positive rates3.

Avidity-based Extracellular Interaction Screening AVEXIS for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions

AVEXIS is a high throughput protein interaction assay developed to systematically screen for novel extracellular receptor-ligand pairs involved in cellular recognition processes. It is specifically designed to detect transient protein interactions that are difficult to identify using other high throughput approaches.

낮은 친화력 세포 수용체 - 리간드 상호 작용의 확장 가능한 감지를위한 욕망 기반의 세포 상호 작용 검사 (AVEXIS)

Extracellular protein:protein interactions between secreted or membrane-tethered proteins are critical for both initiating intercellular communication and ...

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling processes. Extracting sterol-enriched membrane microdomains from plant cells for proteomic analysis is a difficult task mainly due to multiple preparation steps and sources for contaminations from other cellular compartments. The plasma membrane constitutes only about 5-20% of all the membranes in a plant cell, and therefore isolation of highly purified plasma membrane fraction is challenging. A frequently used method involves aqueous two-phase partitioning in polyethylene glycol and dextran, which yields plasma membrane vesicles with a purity of 95% 1. Sterol-rich membrane microdomains within the plasma membrane are insoluble upon treatment with cold nonionic detergents at alkaline pH. This detergent-resistant membrane fraction can be separated from the bulk plasma membrane by ultracentrifugation in a sucrose gradient 2. Subsequently, proteins can be extracted from the low density band of the sucrose gradient by methanol/chloroform precipitation. Extracted protein will then be trypsin digested, desalted and finally analyzed by LC-MS/MS. Our extraction protocol for sterol-rich microdomains is optimized for the preparation of clean detergent-resistant membrane fractions from Arabidopsis thaliana cell cultures.
We use full metabolic labeling of Arabidopsis thaliana suspension cell cultures with K15NO3 as the only nitrogen source for quantitative comparative proteomic studies following biological treatment of interest 3. By mixing equal ratios of labeled and unlabeled cell cultures for joint protein extraction the influence of preparation steps on final quantitative result is kept at a minimum. Also loss of material during extraction will affect both control and treatment samples in the same way, and therefore the ratio of light and heave peptide will remain constant. In the proposed method either labeled or unlabeled cell culture undergoes a biological treatment, while the other serves as control 4.

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Here we describe a robust method for the fractionation of plant plasma membranes into detergent resistant and detergent soluble membranes based on a mixture of unlabeled and in vivo fully 15N labeled Arabidopsis thaliana cell cultures. The procedure is applied for comparative proteomic studies to understand signaling processes.

비교 단백체 분석을위한 신진 대사 라벨링 및 막 분별<em&gt; 애기 장대</em&gt; 서스펜션 세포 배양

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling ...

Capture Compound Mass Spectrometry - A Powerful Tool to Identify Novel c-di-GMP Effector Proteins

Considerable progress has been made during the last decade towards the identification and characterization of enzymes involved in the synthesis (diguanylate cyclases) and degradation (phosphodiesterases) of the second messenger c-di-GMP. In contrast, little information is available regarding the molecular mechanisms and cellular components through which this signaling molecule regulates a diverse range of cellular processes. Most of the known effector proteins belong to the PilZ family or are degenerated diguanylate cyclases or phosphodiesterases that have given up on catalysis and have adopted effector function. Thus, to better define the cellular c-di-GMP network in a wide range of bacteria experimental methods are required to identify and validate novel effectors for which reliable in silico predictions fail.
We have recently developed a novel Capture Compound Mass Spectrometry (CCMS) based technology as a powerful tool to biochemically identify and characterize c-di-GMP binding proteins. This technique has previously been reported to be applicable to a wide range of organisms1. Here we give a detailed description of the protocol that we utilize to probe such signaling components. As an example, we use Pseudomonas aeruginosa, an opportunistic pathogen in which c-di-GMP plays a critical role in virulence and biofilm control. CCMS identified 74% (38/51) of the known or predicted components of the c-di-GMP network. This study explains the CCMS procedure in detail, and establishes it as a powerful and versatile tool to identify novel components involved in small molecule signaling.

The ubiquitous second messenger c-di-GMP controls growth and behavior of many bacteria. We have developed a novel Capture Compound Mass Spectrometry based technology to biochemically identify and characterize c-di-GMP binding proteins in virtually any bacterial species.

캡처 복합 질량 분석 - 소설의 C 디 GMP 이펙터 단백질을 식별 할 수있는 강력한 도구

Considerable progress has been made during the last decade towards the identification and characterization of enzymes involved in the synthesis ...

Non-enzymatic, Serum-free Tissue Culture of Pre-invasive Breast Lesions for Spontaneous Generation of Mammospheres

Breast ductal carcinoma in situ (DCIS), by definition, is proliferation of neoplastic epithelial cells within the confines of the breast duct, without breaching the collagenous basement membrane. While DCIS is a non-obligate precursor to invasive breast cancers, the molecular mechanisms and cell populations that permit progression to invasive cancer are not fully known. To determine if progenitor cells capable of invasion existed within the DCIS cell population, we developed a methodology for collecting and culturing sterile human breast tissue at the time of surgery, without enzymatic disruption of tissue.
Sterile breast tissue containing ductal segments is harvested from surgically excised breast tissue following routine pathological examination. Tissue containing DCIS is placed in nutrient rich, antibiotic-containing, serum free medium, and transported to the tissue culture laboratory. The breast tissue is further dissected to isolate the calcified areas. Multiple breast tissue pieces (organoids) are placed in a minimal volume of serum free medium in a flask with a removable lid and cultured in a humidified CO2 incubator. Epithelial and fibroblast cell populations emerge from the organoid after 10 - 14 days. Mammospheres spontaneously form on and around the epithelial cell monolayer. Specific cell populations can be harvested directly from the flask without disrupting neighboring cells. Our non-enzymatic tissue culture system reliably reveals cytogenetically abnormal, invasive progenitor cells from fresh human DCIS lesions.

Primary cell culture using intact tissue organoids provides a model system that mimics the multi-cellular in vivo microenvironment. We developed a serum-free primary breast epithelium tissue culture model that perpetuates mixed cell culture lineages and exhibits differentiated morphology, without enzymatic tissue disruption. Breast organoids remain viable for &#62;6 months.

비 효소, Mammospheres의 자연 발생에 대한 사전 침습 유방 병변의 혈청이없는 조직 문화

Breast ductal carcinoma in situ (DCIS), by definition, is proliferation of neoplastic epithelial cells within the confines of the breast duct, without ...

Selected Reaction Monitoring Mass Spectrometry for Absolute Protein Quantification

Absolute quantification of target proteins within complex biological samples is critical to a wide range of research and clinical applications. This protocol provides step-by-step instructions for the development and application of quantitative assays using selected reaction monitoring (SRM) mass spectrometry (MS). First, likely quantotypic target peptides are identified based on numerous criteria. This includes identifying proteotypic peptides, avoiding sites of posttranslational modification, and analyzing the uniqueness of the target peptide to the target protein. Next, crude external peptide standards are synthesized and used to develop SRM assays, and the resulting assays are used to perform qualitative analyses of the biological samples. Finally, purified, quantified, heavy isotope labeled internal peptide standards are prepared and used to perform isotope dilution series SRM assays. Analysis of all of the resulting MS data is presented. This protocol was used to accurately assay the absolute abundance of proteins of the chemotaxis signaling pathway within RAW 264.7 cells (a mouse monocyte/macrophage cell line). The quantification of Gi2 (a heterotrimeric G-protein &#945;-subunit) is described in detail.

This protocol describes how to perform absolute quantification assays of target proteins within complex biological samples using selected reaction monitoring. It was used to accurately quantify proteins of the mouse macrophage chemotaxis signaling pathway. Target peptide selection, assay development, and qualitative and quantitative assays are described in detail.

절대 단백질 정량 질량 분석을 모니터링 선정 반응

Absolute quantification of target proteins within complex biological samples is critical to a wide range of research and clinical applications. This ...

Enrichment of Extracellular Matrix Proteins from Tissues and Digestion into Peptides for Mass Spectrometry Analysis

The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins that provides biophysical and biochemical cues that are major regulators of cell proliferation, survival, migration, etc. The ECM plays important roles in development and in diverse pathologies including cardio-vascular and musculo-skeletal diseases, fibrosis, and cancer. Thus, characterizing the composition of ECMs of normal and diseased tissues could lead to the identification of novel prognostic and diagnostic biomarkers and potential novel therapeutic targets. However, the very nature of ECM proteins (large in size, cross-linked and covalently bound, heavily glycosylated) has rendered biochemical analyses of ECMs challenging. To overcome this challenge, we developed a method to enrich ECMs from fresh or frozen tissues and tumors that takes advantage of the insolubility of ECM proteins. We describe here in detail the decellularization procedure that consists of sequential incubations in buffers of different pH and salt and detergent concentrations and that results in 1) the extraction of intracellular (cytosolic, nuclear, membrane and cytoskeletal) proteins and 2) the enrichment of ECM proteins. We then describe how to deglycosylate and digest ECM-enriched protein preparations into peptides for subsequent analysis by mass spectrometry.

This protocol describes a procedure for enriching ECM proteins from tissues or tumors and deglycosylating and digesting the ECM-enriched preparations into peptides to analyze their protein composition by mass spectrometry.

질량 분석기 분석을위한 펩티드로 조직과 소화에서 세포 외 기질 단백질의 농축

The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins that provides biophysical and biochemical cues that are major regulators of ...

Surface Spreading and Immunostaining of Yeast Chromosomes

The small size of nuclei of the budding yeast Saccharomyces cerevisiae limits the utility of light microscopy for analysis of the subnuclear distribution of chromatin-bound proteins. Surface spreading of yeast nuclei results in expansion of chromatin without loss of bound proteins. A method for surface spreading balances fixation of DNA bound proteins with detergent treatment. The method demonstrated is slightly modified from that described by Josef Loidl and Franz Klein1,2. The method has been used to characterize the localization of many chromatin-bound proteins at various stages of the mitotic cell cycle, but is especially useful for the study of meiotic chromosome structures such as meiotic recombinosomes and the synaptonemal complex. We also describe a modification that does not require use of Lipsol, a proprietary detergent, which was called for in the original procedure, but no longer commercially available. An immunostaining protocol that is compatible with the chromosome spreading method is also described.

A method for surface-spreading chromosomes from budding yeast is presented. This method is derived from a method previously described by Loidl and Klein. In addition, we demonstrate a procedure for immunostaining of spread chromosomes.

표면 확산과 효모 염색체의 면역 염색

The small size of nuclei of the budding yeast Saccharomyces cerevisiae limits the utility of light microscopy for analysis of the subnuclear distribution ...

Analyzing Cell Surface Adhesion Remodeling in Response to Mechanical Tension Using Magnetic Beads

Mechanosensitive cell surface adhesion complexes allow cells to sense the mechanical properties of their surroundings. Recent studies have identified both force-sensing molecules at adhesion sites, and force-dependent transcription factors that regulate lineage-specific gene expression and drive phenotypic outputs. However, the signaling networks converting mechanical tension into biochemical pathways have remained elusive. To explore the signaling pathways engaged upon mechanical tension applied to cell surface receptor, superparamagnetic microbeads can be used. Here we present a protocol for using magnetic beads to apply forces to cell surface adhesion proteins. Using this approach, it is possible to investigate not only force-dependent cytoplasmic signaling pathways by various biochemical approaches, but also adhesion remodeling by magnetic isolation of adhesion complexes attached to the ligand-coated beads. This protocol includes the preparation of ligand-coated superparamagnetic beads, and the application of define tensile forces followed by biochemical analyses. Additionally, we provide a representative sample of data demonstrating that tension applied to integrin-based adhesion triggers adhesion remodeling and alters protein tyrosine phosphorylation.

Cell surface adhesions are central in mechanotransduction, as they transmit mechanical tension and initiate the signaling pathways involved in tissue homeostasis and development. Here, we present a protocol for dissecting the biochemical pathways that are activated in response to tension, using ligand-coated magnetic microbeads and force application to adhesion receptors.

자석 구슬을 사용하여 기계 인장에 대한 응답으로 세포 표면 접착 리모델링 분석

Mechanosensitive cell surface adhesion complexes allow cells to sense the mechanical properties of their surroundings. Recent studies have identified both ...