Name: glycopeptide capture for cell surface proteomics
Uploaded: 2014-05-09T20:00:00
Description: Watch this Scientific Journal Video about Glycopeptide Capture for Cell Surface Proteomics at JoVE.com

这项研究已经由西蒙·弗雷泽大学的启动资金支持。

1，嘉实膜<ol><li>添加低渗缓冲液（10mM的Tris-HCl，10mM的氯化钾，1.5毫摩尔MgCl 2，pH 8.0）中与蛋白酶抑制剂混合物到细胞沉淀（〜10 8个细胞），并培育在冰上15-30分钟。裂解细胞通过使样品通过注射器针头（5-10遍），或通过一个Dounce匀浆（15-30冲程）破碎样品。使用血细胞计数器和台盼蓝染色来检查裂解的效率。 </li><li>由裂解物的差速离心获得的微粒体部分在3000 GX 15分钟，然后在...

<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>

在这里，我们介绍一种糖肽捕获策略分析细胞表面蛋白。可以应用该方法来研究分泌蛋白，如血液中，以及在其他体液或细胞培养基中。 该方法的成功依赖于样本的完全消化;因此，消化效率的SDS-PAGE鉴定是必要的，尤其是对于样品的第一次分析。一个完整的消化可具有挑战性的膜蛋白，并可以仅在膜级分的彻底的溶解是可能的。当洗涤剂引入增溶过程开始和尿素的孵育后结?...

细胞表面蛋白与细胞外环境进行交互并从外部中继信号到细胞的内部。因此，这些蛋白，包括细胞外基质蛋白，在细胞生物学和生理学不等的增殖，生长，迁移，分化老化等各方面发挥关键作用。表面蛋白的功能通过与其他细胞，蛋白质和小分子1-3交互。细胞表面蛋白的分子特性是极大的兴趣不仅为生物学家也为制药公司，药品60％以上是针对细胞表面蛋白4。 串联质谱（MS），以其卓越的灵敏度，准确度和吞吐量鉴定蛋白质和多肽，一直是一个强大的工具，用于全球蛋白质组学研究5,6。然而，表面的蛋白质会对质谱为基础的蛋白质组学显著挑战，在低量的重金属修改存在最表面的蛋白质。表面蛋白的跨膜区呈现它们的疏水性;这是特别适用于多通道的跨膜蛋白的情况。因此，这是难以溶解在水溶液中的膜蛋白无洗涤剂的帮助;然而清洁剂的使用普遍抑制高效液相色谱法和质谱1,7,8的蛋白质鉴定的表现。因此，膜蛋白已被鉴定中直接LC-MS基于蛋白质组学很差。 糖基化是发生在细胞表面蛋白9的最重要和最丰富的翻译后修饰之一。聚糖的巨大复杂性和异质性阻碍肽&#39;MS信号10。然而，一些蛋白质组的方法已经使用这个唯一的修改，以丰富的表面蛋白，并从之前的LC-MS分析的蛋白质除去糖部分。这些方法s包括凝集素亲和捕获11和酰肼基或硼酸基化学捕获12以及亲水性色谱分离8,13。聚糖的去除转化膜蛋白常规的蛋白质，并大幅简化了MS表征。因为糖基化还发生在分泌蛋白具有高的溶解度在对比膜蛋白，许多glycoproteomic方法对可溶性蛋白进行了优化，并趋向于具有较低的糖肽的选择性和时被部署到膜蛋白8,14灵敏度。其他的方法也存在丰富，尤其细胞表面蛋白，例如那些使用超速离心15和标记策略16。表征膜蛋白我们的方法和现有的其他方法之间的详细比较了最近进行的17，结果表明，我们的方法可以执行同样出色，如果不是更好，比所有被比较的膜蛋白质组的方法，但具有更高的简易性。 为了帮助研究人员使用这种方法，我们这里详细的一般协议。这种方法综合了现有的glycoproteomics战略几个优点，特别适用于膜糖蛋白构想，但该方法同样适用于分泌蛋白。该方法的特征包括：1）膜蛋白的完全溶解，2）糖肽的糖蛋白，而不是使用固体摄像基板时，以消除潜在的空间位阻的富集，3）使用酰肼化学组成之间的共价键糖肽和捕集基板上，使得键合的糖肽可以容忍严格洗涤的高glycoselectivity，和4）的能力，以对整个捕获过程中一个管用于减少样品损失和缩短操作时间。实施这一方法来研究一个变量后，生物样品包括细胞和组织的ETY，我们观察到的高选择性（&gt; 90％），以糖蛋白8,17,18。

<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

细胞表面蛋白，包括胞外基质蛋白，参与所有主要的细胞过程和功能，例如生长，分化和增殖。这些蛋白质的综合表征为生物标志物的发现，细胞类型识别和药物靶点的选择，以及帮助推动我们的细胞生物学和生理学的了解丰富的信息。表面蛋白，然而，构成，因为它们固有的低丰度，高疏水性，并且重的翻译后修饰显著分析挑战。采取上表面蛋白的糖基化常见的优点，我们在这里介绍一种高通量糖肽捕获方法，集成的现有的几种N-glycoproteomics装置的优点。我们的方法可以丰富从表面的蛋白质所产生的糖肽，并删除其聚糖，使用LC-MS浅显的蛋白质组学研究。该解决N-glycoproteome包括INFormation蛋白质的身份和数量，以及糖基化他们的网站。此方法已被应用到了一系列的研究领域，包括癌症，神经干细胞，和药物毒性。该方法的局限性在于表面的膜蛋白，这样，需要相对大量的样品进行该分析相比，研究集中于胞质蛋白的丰度低。

在实验过程中的代表性流程图总结在图1的标签，并进一步分馏步骤是可选的，细节将在近期公布18描述。另一种方法是分析未修饰的肽，其不与树脂反应。分析所述未修饰的肽的优点包括：非糖基化的肽类和蛋白质，例如在紧密连接claudins的电位标识;一个附加的优点是更准确的定量。基于这些优势，我们称之为这种方法 g lycocapture - 一个ssisted-G的叶形q uantitative...

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

Glycopeptide Capture for Cell Surface Proteomics

细胞表面蛋白的生物学上重要的和广泛的糖基化。我们在这里介绍一个糖肽捕获的方法来溶解，丰富，并且这些去糖基化蛋白质浅显的LC-MS基础的蛋白质组学分析。

糖肽捕获的细胞表面蛋白组学

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

Research

JoVE Journal

Biology

Cell Capture Using a Microfluidic Device

细胞捕获使用一个微流体装置

科研

生物学

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

使用油脂全线Bicelle方法的膜蛋白的高通量结晶

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

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

基于亲和力，外的相互作用筛选（AVEXIS）外低亲和力受体 - 配体相互作用的可扩展检测

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

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

代谢标记和膜分馏的比较蛋白质组学分析<em&gt;拟南芥</em&gt;悬浮细胞培养

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

捕获复合质谱 - 一个强大的工具来确定新型C-二GMP效应蛋白

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

非酶中，预浸润性乳腺癌病变的无血清组织培养的微球体的自然发生

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

选择反应监测质谱法进行绝对定量蛋白质

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

从富集组织及消化细胞外基质蛋白的成肽质谱分析

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

表面扩散和酵母染色体免疫染色

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

分析细胞表面粘附重塑回应机械张力使用磁珠

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