Name: sample preparation for mass-spectrometry-based proteomics analysis of ocular microvessels
Uploaded: 2019-02-22T14:30:00
Description: Watch this Scientific Journal Video about Sample Preparation for Mass-spectrometry-based Proteomics Analysis of Ocular Microvessels at JoVE.com

manicam 博士得到了美因法约翰内斯古腾堡大学医学中心的内部大学研究资助 (stufe 1) 和德国 mforschungsgemeinschaft 的资助 (ma 800 6/1-1) 的资助。

所有使用动物样本的实验程序都是严格按照视力和眼科研究协会 (arvo) 《关于在眼科和视力研究中使用动物的声明》和机构指南进行的。这项研究是在美因茨大学医疗中心眼科进行和批准的。
请注意:猪的眼睛与视神经和眼外组织是新鲜的从当地屠宰场立即死亡。用冰凉磷酸盐缓冲盐水 (pbs) 将有钙化的眼睛输送到实验室, 并立即使用。所使用的工作流的示意图概述...

<ol>
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对各种眼样本进行全面的蛋白质组分析是阐明健康和疾病中涉及的分子机制和信号通路的重要和不可或缺的第一步。为了获得高质量的数据并确保从这些分析中获得的结果的重现性, 前面的样品制备步骤至关重要, manal 等人在深入讨论样品处理程序的审查中强调了这一点。对于眼睛的不同部位采用二维凝胶电泳和质谱策略 1。在这些研究中, 我们目前的研究提供了一个优化的逐步?...

蛋白质组学领域的进步, 使综合和无与伦比的数据收集能力, 极大地改变了我们对某些疾病条件背后的分子机制的理解, 以及在反映特定细胞群或组织的生理状态1、2、3、4。蛋白质组学也被证明是眼科研究的一个重要平台, 因为对不同的眼睛样本进行了敏感性和无偏见的分析, 有助于确定潜在的疾病标志物, 以便最终诊断和预后。近年来的许多研究都很有证明, 包括我们的一些研究 1、5、6、7、8、9、10.然而, 由于伦理原因, 通常很难获得用于蛋白质组学分析的人体样本, 特别是考虑到需要健康的个人提供控制材料进行可靠的比较分析。另一方面, 获得足够数量的样品以进行最佳和可靠的质谱分析也具有挑战性。这对于数量有限的生物材料, 如眼睛的微血管尤其重要。其中一个主要的反流血管, 发挥调节眼血流的关键作用是短后睫毛动脉 (scca)。这个维管床上的任何扰动或异常都可能导致严重的临床影响, 从而导致青光眼和非动脉前缺血性视神经病变 (naion)等几种有视觉症状的疾病的发病机制11,12. 然而, 由于上述缺点, 缺乏阐明该动脉床蛋白质组变化的研究。因此, 近年来, 由于人类和猪13之间的形态和系统发育相似性很高, 家猪 (家养林奈, 1758) 已成为眼科研究中的一个很好的动物模型 。 14,15。猪眼样本很容易获得, 最重要的是, 更准确地表示人体组织。
考虑到这些血管在眼睛中的重要作用, 以及缺乏有效提取蛋白质的方法和从这些微血管中分析的方法, 我们以前已经用内部的方法对猪 pca 的蛋白质组进行了表征。协议, 导致识别了大量的蛋白质16。在这项研究的基础上, 我们在本文中进一步优化和深入地描述了我们的方法, 允许使用猪 scca 作为模型组织从微小数量的样本中进行蛋白质组分析。尽管这项研究的主要目的是建立一个 ms 兼容的方法, 质量有限的眼血管, 我们提供了大量的实验证据, 所述的工作流程也可以广泛地适用于各种组织为基础的样本。
根据设想, 这一工作流程将有助于从少量材料中制备高质量的 ms 兼容样品, 以便进行全面的蛋白质组分析。

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			<td height="21" style="height:21px;width:436px;">A. Chemicals</td>
			<td style="width:281px;"></td>
			<td style="width:120px;"></td>
			<td style="width:203px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1, 4-Dithiothreitol (DTT)</td>
			<td>Sigma-Aldrich</td>
			<td style="width:120px;">1.11474</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ammonium bicarbonate (ABC, CH&#8325;NO&#8323;)</td>
			<td>Sigma-Aldrich</td>
			<td>5.33005</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Calcium chloride dihydrate (CaCl2)&#160;</td>
			<td>&#160;Carl Roth&#160;</td>
			<td>5239.1</td>
			<td>2.5 mM&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dulbecco&#39;s phosphate-buffered saline (PBS)&#160;</td>
			<td style="width:281px;">Thermo Fisher Scientific</td>
			<td>14190169</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Formic acid (CH2O2)</td>
			<td style="width:281px;">AppliChem</td>
			<td style="width:120px;">A0748</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">HPLC-grade acetonitrile (ACN, C2H3N)</td>
			<td style="width:281px;">AppliChem</td>
			<td style="width:120px;">A1605</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">HPLC-grade methanol (CH3OH)</td>
			<td style="width:281px;">Fisher Scientific</td>
			<td style="width:120px;">M/4056/17</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HPLC-grade water&#160;</td>
			<td style="width:281px;">AppliChem</td>
			<td style="width:120px;">A1589</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Iodoacetamide (IAA)</td>
			<td>Sigma-Aldrich</td>
			<td style="width:120px;">I6125</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Kalium chloride (KCl)&#160;</td>
			<td>&#160;Carl Roth&#160;</td>
			<td>6781.1</td>
			<td>4.7 mM&#160;</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Kalium dihydrogen phosphate (KH2PO4)</td>
			<td>&#160;Carl Roth&#160;</td>
			<td>3904.2</td>
			<td>1.2 mM&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LC-MS-grade acetic acid</td>
			<td style="width:281px;">&#160;Carl Roth&#160;</td>
			<td style="width:120px;">AE69.1</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Magnesium sulphate (MgSO4)&#160;&#160;</td>
			<td>&#160;Carl Roth&#160;</td>
			<td style="width:120px;">261.2</td>
			<td>1.2 mM&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NuPAGE Antioxidant</td>
			<td style="width:281px;">Thermo Fisher Scientific (Invitrogen)</td>
			<td style="width:120px;">NP0005</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NuPAGE LDS Sample buffer&#160;</td>
			<td style="width:281px;">Thermo Fisher Scientific (Invitrogen)</td>
			<td>NP0007</td>
			<td>4x</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NuPAGE MES SDS Running Buffer&#160;</td>
			<td style="width:281px;">Thermo Fisher Scientific (Invitrogen)</td>
			<td>NP0002</td>
			<td>20x</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NuPAGE Sample reducing agent&#160;</td>
			<td style="width:281px;">Thermo Fisher Scientific (Invitrogen)</td>
			<td>NP0004</td>
			<td>10x</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SeeBlue Plus2 pre-stained protein standard&#160;</td>
			<td style="width:281px;">Thermo Fisher Scientific (Invitrogen)</td>
			<td style="width:120px;">LC5925</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sequencing grade modified trypsin</td>
			<td style="width:281px;">Promega</td>
			<td style="width:120px;">V5111</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium chloride (NaCl)</td>
			<td>&#160;Carl Roth&#160;</td>
			<td>9265.2</td>
			<td>118.3 mM&#160;</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Sodium hydrogen carbonate (NaHCO3)</td>
			<td>&#160;Carl Roth&#160;</td>
			<td>965.3</td>
			<td>25 mM&#160;</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Trifluoroacetic acid (TFA,&#160; C2HF3O2)</td>
			<td style="width:281px;">Merck Millipore</td>
			<td style="width:120px;">108178</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#945;-(D)-(+)- Glucose monohydrate</td>
			<td>&#160;Carl Roth&#160;</td>
			<td>6780.1</td>
			<td>11 mM&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">B. Reagents and Kits</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.5mm zirconium oxide beads&#160;</td>
			<td style="width:281px;">Next Advance</td>
			<td style="width:120px;">ZROB05</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1.0mm zirconium oxide beads&#160;</td>
			<td style="width:281px;">Next Advance</td>
			<td style="width:120px;">ZROB10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Colloidal Blue Staining&#160; Kit</td>
			<td style="width:281px;">Thermo Fisher Scientific (Invitrogen)</td>
			<td style="width:120px;">LC6025</td>
			<td>To stain 25 mini gels per kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NuPAGE 4-12 % Bis-Tri gels</td>
			<td>Thermo Fisher Scientific (Invitrogen)</td>
			<td>NP0321BOX</td>
			<td>1.0 mm, 10-well</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pierce Bicinchoninic Acid (BCA) Protein Assay Kit&#160;</td>
			<td style="width:281px;">Thermo Fisher Scientific</td>
			<td style="width:120px;">23227</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ProteoExtract Transmembrane Protein Extraction Kit, TM-PEK</td>
			<td style="width:281px;">Merck Millipore</td>
			<td style="width:120px;">71772-3</td>
			<td>20 reactions per kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tissue Protein Extraction Reagent (T-PER)</td>
			<td style="width:281px;">Thermo Scientific</td>
			<td style="width:120px;">78510</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">C. Tools</td>
			<td style="width:281px;"></td>
			<td style="width:120px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">96-well V-bottom plates</td>
			<td style="width:281px;">Greiner Bio-One</td>
			<td style="width:120px;">651180</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Corning 96-well flat-bottom plates</td>
			<td style="width:281px;">Sigma-Aldrich</td>
			<td style="width:120px;">CLS3595-50EA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Disposable microtome blades</td>
			<td>pfm Medical</td>
			<td>207500014</td>
			<td></td>
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			<td height="21" style="height:21px;">Disposable scalpels #21</td>
			<td>pfm Medical</td>
			<td>200130021</td>
			<td></td>
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			<td height="21" style="height:21px;">Dissection pins&#160;</td>
			<td style="width:281px;">Carl Roth</td>
			<td style="width:120px;">PK47.1</td>
			<td></td>
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			<td height="21" style="height:21px;">Extra Fine Bonn Scissors</td>
			<td style="width:281px;">&#160;Fine Science Tools</td>
			<td style="width:120px;">14084-08</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Falcon conical centrifuge tubes (50 mL)</td>
			<td>Fisher Scientific</td>
			<td>14-432-22</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mayo scissors, Tough cut</td>
			<td style="width:281px;">&#160;Fine Science Tools</td>
			<td style="width:120px;">14130-17</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Precision tweezers</td>
			<td style="width:281px;">&#160;Fine Science Tools</td>
			<td>11251-10</td>
			<td>Type 5</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Precision tweezers, straight with extra fine tips</td>
			<td style="width:281px;">Carl Roth</td>
			<td>LH53.1</td>
			<td>Type 5</td>
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			<td style="width:281px;">Ratiolab (vWR)</td>
			<td style="width:120px;">RATI6018412</td>
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			<td height="21" style="height:21px;">Standard pattern forceps</td>
			<td style="width:281px;">&#160;Fine Science Tools</td>
			<td style="width:120px;">11000-12</td>
			<td></td>
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			<td height="21" style="height:21px;">Student Vannas spring scissors</td>
			<td>&#160;Fine Science Tools</td>
			<td>91501-09</td>
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			<td style="width:281px;">Geuder</td>
			<td style="width:120px;">19760</td>
			<td>&#160;Straight, 77 mm</td>
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			<td height="25" style="height:25px;">ZipTipC18 pipette tips</td>
			<td style="width:281px;">Merck Millipore</td>
			<td>ZTC18S096</td>
			<td></td>
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			<td style="width:120px;"></td>
			<td></td>
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			<td style="width:281px;">Thermo Scientific, Rockford, USA</td>
			<td style="width:120px;">72105-150565</td>
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			<td style="width:281px;">Thermo Scientific, Rockford, USA</td>
			<td style="width:120px;">72105-030515</td>
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			<td style="width:120px;">UFC500396</td>
			<td>Pack of 96.</td>
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			<td>Sartorius</td>
			<td>H51</td>
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			<td style="width:120px;">HTS Pal</td>
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			<td style="width:120px;">NA-BB-25</td>
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			<td height="21" style="height:21px;">Eppendorf concentrator, model 5301</td>
			<td style="width:281px;">Sigma-Aldrich</td>
			<td style="width:120px;">Z368172</td>
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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Eppendorf microcentrifuge, model 5424</td>
			<td style="width:281px;">Fisher Scientific</td>
			<td style="width:120px;">05-403-93</td>
			<td>Non-refrigerated</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Heraeus Primo R Centrifuge</td>
			<td style="width:281px;">Thermo Scientific</td>
			<td style="width:120px;">75005440</td>
			<td>Refrigerated</td>
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			<td height="21" style="height:21px;">Labsonic M Ultrasonic homogenizer&#160;</td>
			<td style="width:281px;">Sartorius</td>
			<td style="width:120px;">BBI-8535027</td>
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		<tr height="21">
			<td height="21" style="height:21px;">LC-MS pump, model Rheos Allegro</td>
			<td style="width:281px;">Thermo Scientific, Rockford, USA</td>
			<td style="width:120px;">22080</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">LTQ Orbitrap XL mass spectrometer&#160;</td>
			<td>Thermo Scientific, Bremen, Germany</td>
			<td style="width:120px;"></td>
			<td></td>
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			<td height="21" style="height:21px;">Multiskan Ascent plate reader&#160;</td>
			<td style="width:281px;">Thermo Labsystems</td>
			<td style="width:120px;">v2.6</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Rotator with vortex&#160;</td>
			<td style="width:281px;">neoLab</td>
			<td style="width:120px;">7-0045</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Titanium probe (&#216; 0.5mm, 80mm long)</td>
			<td style="width:281px;">Sartorius</td>
			<td style="width:120px;">BBI-8535612</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ultrasonic bath, type RK 31</td>
			<td style="width:281px;">Bandelin</td>
			<td style="width:120px;">329</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Xcell Surelock Mini Cell</td>
			<td>Life Technologies</td>
			<td>El0001</td>
			<td></td>
		</tr>
	</tbody>
</table>

sample preparation for mass-spectrometry-based proteomics analysis of ocular microvessels

利用体外孤立的眼血管, 用先进的技术方法破译眼睛的病理生理状态, 极大地扩大了我们对某些疾病的认识。质谱 (ms) 的蛋白质组学已成为一个强有力的工具, 以解开改变分子机制和蛋白质信号通路的血管床在健康和疾病。然而, 在 ms 分析之前的样品制备步骤对于获得可重现的结果和深入阐明复杂蛋白质组至关重要。这对于眼科微血管的制备尤其重要, 因为在眼科微血管中, 可用于分析的样品数量往往有限, 因此对最佳蛋白质提取构成了挑战。本文试图提供一个有效的, 快速和可靠的方案, 样品制备从一个示范后腰椎眼血管床使用猪短后睫毛动脉。本方法的重点是从样品的上清液和颗粒的蛋白质提取程序后, 均质化, 样品清洗前的离心过滤装置和肽纯化在液相色谱-电喷雾电离-线性离子陷阱-轨道记录 ms 系统中的无标签量化步骤。虽然这种方法是专门为眼科微血管的蛋白质组学分析开发的, 但我们也提供了令人信服的证据, 证明它也可以很容易地用于其他组织基样品。

样本有限是眼科研究的主要缺陷之一。相应地, 从少量样本 (如眼睛血管) 中提取最佳蛋白质产量的方法也常常值得商榷。到目前为止, 特别是从反疣血管中提取蛋白质的方法很少。因此, 作为方法优化的第一步, 并作为一项原则证明, 将几种常用的蛋白质提取剂的疗效和鲁棒性与相对较新的试剂 t-per 进行了试点研究, 并利用心脏组织进行了研究小鼠的情况 (由于优化步骤的样...

Watch this Scientific Journal Video about Sample Preparation for Mass-spectrometry-based Proteomics Analysis of Ocular Microvessels at JoVE.com

Sample Preparation for Mass-spectrometry-based Proteomics Analysis of Ocular Microvessels

眼微血管床的蛋白质组特征是深入了解人类许多眼部病理的关键。本研究展示了一种有效、快速、稳健的方法, 用于利用猪短的后睫毛动脉作为基于质谱的蛋白质组学分析的模型血管, 从小血管中提取蛋白质和制备样品。

基于性光谱的眼微血管蛋白质组学样品制备

The use of isolated ocular blood vessels in vitro to decipher the pathophysiological state of the eye using advanced technological approaches has greatly ...

制备高质量的质谱学兼容样品，对眼样进行全面的蛋白质组分析，对于阐明与健康和疾病有关分子机制和信号通路至关重要。所述工作流程代表一种简单而稳健的方法，用于严格样品制备步骤，专门用于分析质量有限的样品，如眼微血管。虽然这项研究的主要目的是建立一个与MS兼容的眼血管方法，但所述工作流程可以广泛应用于各种基于细胞和组织的样本。一般来说，对这种方法的新鲜人可能难以辨认，因为识别和隔离选定的短后侧动脉或SPCA分支可能具有挑战性。新鲜猪眼，连同视神经和外眼组织，立即从当地的屠宰场获得验尸。将眼球放在含有冰冷克雷布斯-亨塞莱特缓冲液的解剖室中。用一把锋利的梅奥剪刀小心地切开周围的肌肉和组织。用手术刀做切口，用一把锋利的梅奥剪刀沿着赤道平面切割地球，直到眼睛被分成前半部分和后半部分。使用一对标准图案钳从眼睛的后半部分取出尽可能多的玻璃体。使用解剖针小心地固定眼睛的后半部分，轻轻切开视神经周围的结缔组织，用一双学生Vannas弹簧剪刀露出潜在的逆光杆。用一对五型精密钳子和Vannas帽切除术剪刀将伞状和侧短后侧动脉与周围的结缔组织隔离开来。在冰冷PBS中冲洗隔离动脉以去除污染物和血液残留物之前，使用额外的细尖钳和剪刀轻轻去除动脉部分的结缔组织。从两只眼睛汇集动脉，获得一个生物复制，然后使用分析平衡称量样本。在每个管上，加入0.5和1毫米氧化硅珠的混合物，然后加入组织蛋白提取试剂。现在，将样品管装入搅拌机均质器中。将计时器设置为两分钟，将速度级别设置为 6，并开始均质化。运行后，检查样品完全均质，并在每次运行之间将样品留在冰上。重复循环，直到样品完全均质。小心移液器均质化成新鲜的微离心管。将样品在10，000次G下离心20分钟，在4摄氏度下产生不溶性蛋白质。小心移液含有可溶性蛋白质的上清液，无需接触颗粒层，并转移到新鲜的微离心管中。在每个颗粒样品中加入200微升的萃取缓冲液2A和5微升蛋白酶抑制剂鸡尾酒。然后，用移液器多次悬浮颗粒。使用超声波均质器使颗粒完全均质。将振幅设置为 60，然后循环设置为 1。使用钛制成的探针，该探针适用于体积小的样品的均质化。将探针浸入冰的颗粒和萃取缓冲液混合物中，然后按下启动按钮。对样品进行声波化，直到颗粒团完全均匀，每次声波两次之间暂停几秒钟。目视检查完全均质。将均质与移液器混合几次，以确保没有团块。使用离心过滤装置，具有三千克当吨截止时间。将三千龙的截止过滤器单元插入微离心管中。将200微升样品均质添加到一个过滤装置中，并将200微升的去维化水加入同一过滤器。安全封盖后，将过滤装置放入离心机中，在4摄氏度下旋转14，000次G，旋转15分钟。离心后，将含有样品浓缩物的过滤装置与含有滤液的微离心管分离，丢弃滤液。通过将400微升的去化水加入过滤单元来重组浓缩物。重复过滤三次后，小心地将清洁的样品浓缩物移液到干净的微管中。准备文本协议中列出的一维凝胶电泳样本，并很好地与移液器混合。在干块加热器中加热样品 70 摄氏度 15 分钟，冷却至室温。使用移液器，以及预着色的蛋白质标准作为分子质量标记，小心地加载每车道 50 微克样品。在 175 伏的恒定电压下运行凝胶约 60 分钟。在运行结束时，使用凝胶刀小心地从盒式板中去除凝胶，然后将凝胶转移到凝胶染色盒中。执行凝胶的固定和染色，如文本协议中所述。摇动染色溶液中的凝胶过夜，以获得最佳的整体效果。小心地将染色溶液去掉，然后用200毫升的去维化水代替，然后在水中摇动凝胶至少7至8个小时以清除背景。使用干净的新微切片将蛋白质带从凝胶中切除。将带子切成小块，小心地将凝胶片转移到1.5毫升微离心管中。加入含有100毫摩尔碳酸氢铵和醋酸酯的500微升脱盐溶液。在室温下孵育样品30分钟，偶尔摇晃。小心地移液出脱污溶液。目视检查是否有残留污渍的管，如果凝胶片仍为蓝色染色，请重复此步骤。加入约400微升新鲜准备的DTT溶液，并在56摄氏度下孵育30分钟。使用移液器丢弃还原溶液后，加入约 400 微升新鲜准备的 IAA 溶液，并在室温的黑暗中孵育 30 分钟。用移液器拆下alkylating缓冲液并丢弃。在室温下加入500微升整齐的乙酰酸盐10至15分钟，直到凝胶片收缩并变得不透明。将乙酰丙酮移出，在发动机罩下风干凝胶片 5 至 10 分钟。然后，移液器50微升的三辛溶液进入每个管，以完全覆盖凝胶片，并在4摄氏度下孵育管。30 分钟后，添加足够体积的 trypsin 缓冲液，如有必要，可完全覆盖凝胶片。在37摄氏度下孵育样品过夜。要执行肽提取，请小心地将提取的肽溶液从管子中移液，并转移到干净的微管中。在离心真空蒸发器中干燥上一个液。将100微升的萃取缓冲液加入每个管中，用凝胶片进行孵化30分钟。将上一液移入同一微管中，根据各自的带子，将提取的肽放入相同的微管中，并在真空离心机中干燥。按照文本协议中所述，继续进行肽纯化和液相色谱-电喷雾电离MS/MS分析。此处描述了用每种洗涤剂提取的样品中的蛋白质总量。产量最高的来自用组织蛋白提取缓冲液提取的组织，其次是DDM、CHAPS、ASB-14，以及从ACN和TFA中提取的最低产量。一致地，所鉴定的总蛋白质也是组织蛋白提取缓冲液提取样品中最高的，与总产量的趋势相同。此处显示的是 SPCA 的一维凝胶电泳蛋白配置文件的比较，在经过优化的样品制备和清洁步骤之前和之后。总体而言，在第三车道观察到蛋白质带的涂片度高和分离不良。此配置文件表明，样品可能含有萃取试剂，以及污染物，如脂质和细胞碎片。然而，SPCA样品被分离成上经剂和颗粒，然后根据优化的协议，导致模范的一维凝胶电泳轮廓。从眼微纤维中提取快速、稳健、高效的蛋白质的优化方法也可以很容易地应用于其他基于组织的样品中。此处显示的是上清液的蛋白质图和大脑中和心脏组织样本的颗粒。在尝试此程序时，重要的是要记住使样品完全均质化，将上清液与颗粒分离，并去除污染物和萃取试剂。该技术开发后，为实验和转化眼科领域的研究人员以猪眼模型为模型，彻底探索眼血管的蛋白质组铺平了道路。

Research

JoVE Journal

Biology

Western Blotting: Sample Preparation to Detection

免疫印迹检测样品制备

Western blotting is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. It uses gel ...

科研

生物学

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 ...

Proteomic Sample Preparation from Formalin Fixed and Paraffin Embedded Tissue

修正了福尔马林蛋白质组学样品的制备和石蜡包埋组织

Preserved clinical material is a unique source for proteomic investigation of human disorders. Here we describe an optimized protocol allowing large scale ...

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 ...

Sample Preparation for Mass Cytometry Analysis

样品制备质谱术分析

Mass cytometry utilizes antibodies conjugated with heavy metal labels, an approach that has greatly increased the number of parameters and opportunities ...

An Alternative Approach for Sample Preparation with Low Cell Number for TEM Analysis

对于样品制备与TEM分析低细胞数量的另一种方法

Transmission electron microscopy (TEM) provides details of the cellular organization and ultrastructure. However, TEM analysis of rare cell populations, ...

Sample Preparation and Imaging of Exosomes by Transmission Electron Microscopy

透射电镜下体的样品制备与成像

Exosomes are nano-sized extracellular vesicles secreted by body fluids and are known to represent the characteristics of cells that secrete them. The ...

Miniaturized Sample Preparation for Transmission Electron Microscopy

透射电镜的小型化样品制备方法

Due to recent technological progress, cryo-electron microscopy (cryo-EM) is rapidly becoming a standard method for the structural analysis of protein ...

Comprehensive Workflow of Mass Spectrometry-based Shotgun Proteomics of Tissue Samples

组织样品质谱基猎枪蛋白造影学的综合工作流程

Recent advances in mass spectrometry have resulted in deep proteomic&#160;analysis along with the generation of&#160;robust and&#160;reproducible ...

Sample Preparation for Metabolic Profiling using MALDI Mass Spectrometry Imaging

使用 MALDI 质谱成像进行代谢分析的示例准备

Metabolomics, the study to identify and quantify small molecules and metabolites present in an experimental sample, has emerged as an important tool to ...