Name: sample preparation for endopeptidomic analysis in human cerebrospinal fluid
Uploaded: 2017-12-04T13:00:00
Description: Watch this Scientific Journal Video about Sample Preparation for Endopeptidomic Analysis in Human Cerebrospinal Fluid at JoVE.com

非常感谢 Tanveer Batth 和同事在建立预分馏法方面的建议。
这项工作得到了瑞典研究理事会、Wallström 和 Sjöblom 基金会、枪支和 Bertil Stohne 基金会 Stiftelse、马格努斯 Bergwall 基金会、Åhlén 基金会、Alzheimerfonden、Demensförbundet、Stiftelsen för 的资助。格姆拉斯坦 Tjänarinnor, 克努特和爱丽丝的基础, Frimurarestiftelsen, 和 Västra Götalandsregionen。
这个项目的主要资助对象是 Kaj Blennow, 亨利 Zetterberg 和约翰 Gobom。

下面所述的协议是在以前的研究中使用的一个精炼版本, 其中大量的内源性多肽在人类 CSF15中被识别出来。对原始协议的更新包括对脑脊液化学预处理的细微改动, 以及用于离线高 pH 反相高效 HPLC 预分馏的梯度优化。
道德考虑
所有关于瑞典病人和控制材料的研究都得到了伦理委员会的批准: St. 约兰·佩尔松 (文献 2005-554-31/3)?...

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采用高效液相色谱法预分馏步骤, 采用分子量超滤法回收内源性多肽11降低了相对样本复杂度, 从而使5倍大要研究的样本量。这反过来增加了每个分数中所含的肽子集的浓度, 从而提高了检测低丰肽的几率。
通过对三种蛋白质组软件并行使用的内源肽进行识别, 可以扩大已知的脑脊液 endopeptidome 超过10倍。共16395内源性多肽在一个合并脑脊液标本的初步试验中?...

脑脊液中的生物标志物目前正转化为神经退行性疾病的研究。在阿尔茨海默氏病, 最常见的神经退行性疾病, 影响超过6000万人的全球1,2, 一个生物标记三重组成的肽淀粉样β, 微管稳定蛋白 tau, 和磷酸化 tau 形态, 能检测出高灵敏度和特异性的疾病, 并已纳入诊断研究标准3。在其他神经退行性疾病, 如帕金森氏病和多发性硬化症, 蛋白质组研究发现了许多生物标记候选, 其中一些目前正在评估临床研究4,5 ,6。
除了蛋白质, CSF 还含有丰富的内源性多肽7,8,9,1011.这些肽构成了许多脑源性蛋白质的裂解产物, 同时也代表了一种潜在的重要的疾病生物标志物来源。为了增加人类脑脊液中已识别的内源性多肽的库存, 并在临床研究中进行 csf endopeptidomic 分析, 开发了用于样品制备和 LC-MS 分析的方法 (在图 1中包含了一个简短的协议方案).该方法在最近的一项研究中的应用, 导致了来自几个非特定诊断个体的汇集脑脊液样本中的近16400内源性脑脊液肽的鉴定, 扩大已知 csf endopeptidome 十倍13。该方法可选择与等压标签法结合使用, 以量化。
样品制备
脑脊液中蛋白质质量的主要来源是血浆成分 (如白蛋白和免疫球蛋白) 通过血脑屏障14,15。它们的高丰度阻碍了对低丰度脑源样件的检测。内源性肽可以很容易地与高丰度的蛋白质分离, 从而允许大量的脑脊液肽提取物用于 LC-MS 分析, 从而能够检测到低丰度的肽。
在这里提出的协议中, 分子量的分子过滤是用来分离脑脊液肽的蛋白质部分;已在以前的几项研究中使用的方法8、9、10、11、12、16。过滤步骤之后是一个离线反相高效液相色谱预分馏步骤, 在高 pH 移动相位梯度。通过串联两个反相 HPLC 步骤, 以 pH 值为主要区别, 两个步骤的选择性差异主要是由于不同的肽电荷状态而导致的多肽潴留。在酸性条件下, 高 pH 肽预分馏在 LC-MS 的应用在增加肽识别17,18, 甚至是在复杂生物的优势样品比较正交分离模式19, 如强 cat 离子交换 (SCX) 和 RP20。为了缩短分析时间, 使用了串联方案, 将每12个th分数 (例如、分数1、13、25、37和 49) 汇集在一起, 由于反相高效色谱法的高分辨力, 仍然很大程度上避免了不同的肽的共洗LC-MS 步骤20,21中的分数。
肽鉴定
peptidomic 研究中的肽鉴定与蛋白质组学研究不同, 因为在数据库搜索中不能指定酶解理, 因此, 识别率通常低于11。最近的一项研究13表明, Sequest 和吉祥物获得的内源性多肽的识别率在使用自适应评分法修改相应软件程序的默认评分算法时得到了显著改善算法过滤, 表明内源肽的最佳评分算法与胰肽13不同。在这项研究中, 基于自动肽从头测序使用软件峰值 (BSI) 被发现是互补的两个片段离子指纹为基础的搜索引擎, 导致了显着更大的一套识别肽.

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	<tbody>
		<tr height="38">
			<td height="38" style="height:38px;width:547px;">&#160;1 M Triethylammonium bicarbonate</td>
			<td style="width:339px;">Fluka, Sigma-Aldrich</td>
			<td style="width:339px;">17902-100ML</td>
			<td style="width:339px;">TEAB</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">&#160;8 M Guanidinium hydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>G7294-100ML</td>
			<td>GdnHCl</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:547px;">Tris(2-carboxyethyl)phosphine hydrochloride</td>
			<td>Pierce</td>
			<td>20490</td>
			<td>TCEP</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">Iodoacetamide</td>
			<td>SIGMA</td>
			<td>I1149-5G</td>
			<td>IAA</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">Hydroxylamine 50% (w/w)</td>
			<td>Sigma-Aldrich</td>
			<td>457804-50ML</td>
			<td></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">Acetonitrile, Far UV, HPLC gradient grade</td>
			<td>Sigma-Aldrich</td>
			<td>271004-2L</td>
			<td>AcN</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">Formic acid</td>
			<td>Fluka, Sigma-Aldrich&#160;</td>
			<td>56302-1mL-F</td>
			<td>FA</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">Triflouroacetic acid</td>
			<td>Sigma-Aldrich</td>
			<td>T6508-10AMP</td>
			<td>TFA</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">Ammonium hydroxide solution</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>30501-1L-1M</td>
			<td>NH4OH</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">Amicon Ultra-15 Centrifugal Filter Unit with Ultracel-30 membrane</td>
			<td>Merck Millipore</td>
			<td>UFC903024</td>
			<td>MWCO-filter</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">Sep-Pak C18, 100 mg</td>
			<td>Waters</td>
			<td>WAT023590</td>
			<td>SPE-column</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">Resprep 12-port SPE Manifold&#160;</td>
			<td>Restek</td>
			<td>26077</td>
			<td>Vacuum manifold</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">TMT10plex Isobaric Label Reagent Set</td>
			<td>Thermo Fisher Scientific</td>
			<td>90110</td>
			<td>TMT10plex</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">UltiMate 3000 RSLCnano LC System</td>
			<td>Dionex</td>
			<td>5200.0356</td>
			<td>Online sample separation</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">Ultimate 3000 RPLC Rapid Separation Binary System</td>
			<td>Dionex</td>
			<td>IQLAAAGABHFAPBMBEZ</td>
			<td>Offline high-pH fractionation</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">Orbitrap Fusion Tribrid mass spectrometer</td>
			<td>Thermo Scientific</td>
			<td style="width:339px;">IQLAAEGAAPFADBMBCX</td>
			<td>Mass spectrometer for sample analysis</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">Proteome Discoverer 2.0&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>IQLAAEGABSFAKJMAUH</td>
			<td>Proteomics search platform</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">Mascot v2.4</td>
			<td>Matrix Science</td>
			<td style="width:339px;">&#160;-&#160;</td>
			<td>Proteomics search engine</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">Sequest HT</td>
			<td>Thermo</td>
			<td style="width:339px;">&#160;-&#160;</td>
			<td>Proteomics search engine</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">PEAKS v7.5&#160;</td>
			<td>Bioinformatic Solutions Inc.)</td>
			<td style="width:339px;">&#160;-&#160;</td>
			<td>Proteomics search engine</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">Acclaim PepMap 100, 75 &#181;m x 2 cm, C18, 100 &#197; pore size, 3 &#181;m particle size</td>
			<td>Thermo Fisher Scientific</td>
			<td>164535</td>
			<td>Trap column (nano HPLC)</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">Acclaim PepMap C18, 75 &#181;m x 500 mm, 100&#197; pore size,&#160;2 &#181;m particle size</td>
			<td>Thermo Fisher Scientific</td>
			<td>164942</td>
			<td>Separation Column (nano HPLC)</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">Savant SpeedVac High Capacity Concentrators</td>
			<td>Thermo Fisher Scientific</td>
			<td>SC210A-230</td>
			<td>SpeedVac/Vacuum concentrator</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">XBridge Peptide BEH C18 Column, 130&#197;, 3.5 &#181;m, 2.1 mm X 250 mm</td>
			<td>Waters</td>
			<td>186003566</td>
			<td>Separation Column (micro HPLC)</td>
		</tr>
	</tbody>
</table>

sample preparation for endopeptidomic analysis in human cerebrospinal fluid

本协议描述了一种用于识别人脑脊液中内源性多肽的方法。为此, 本文提出了一种基于分子量分子过滤和质谱分析的方法, 并结合离线高 pH 反相高效液相色谱预分馏步骤。
脑脊液分泌是去除中枢神经系统细胞分子的主要途径。因此, 中枢神经系统中的许多过程都反映在脑脊液中, 使它成为一种有价值的诊断液。CSF 有一个复杂的组成, 包含的蛋白质的浓度范围为 8-9 的数量级。除了蛋白质, 以前的研究还表明存在大量的内源性多肽。虽然不像蛋白质那样广泛研究, 但它们也可能作为生物标志物而具有潜在的兴趣。
内源性肽通过分子过滤与脑脊液蛋白含量分离。通过去除样品中的大多数蛋白质含量, 可以增加研究的样品体积, 从而也能提高内源性多肽的总量。在 LC-质谱分析之前, 通过在碱性 pH 值上采用反相 (RP) HPLC 预分馏步骤, 解决了过滤肽混合物的复杂性。分馏与一个简单的串联方案结合在一起, 其中60个分数被汇集到 12, 分析时间消耗量可以从而减少, 当仍然主要避免 co 洗脱。
通过使用三种不同的肽/蛋白质识别软件程序, 并结合结果进行了自动肽鉴定。不同的方案是互补的, 而不是可比的, 少于15% 的标识重叠之间的三。

本文介绍的方法已在三项研究中应用和评价前的样品前分馏 (表 1)。第一项研究使用离线 LC 在 MALDI 靶板上发现脑脊液分数, 并导致730已确定内源性肽11。在以下两项研究中, 使用了等压标签。主要在病例/对照研究中, 对 CSF endopeptidome 和蛋白质组中的潜在生物标志物进行鉴定和描述, 同时24, 并在第二项研究中使用等压标记用于监测治疗效果体内γ-分泌抑制?...

Watch this Scientific Journal Video about Sample Preparation for Endopeptidomic Analysis in Human Cerebrospinal Fluid at JoVE.com

Sample Preparation for Endopeptidomic Analysis in Human Cerebrospinal Fluid

提出了一种人脑脊液内源性多肽的质谱分析方法。通过采用分子量切断过滤、色谱预分馏、质谱分析以及随后的多肽识别策略组合, 就可以将已知的脑脊液 peptidome 扩大近十倍以前的研究。

人脑脊液 Endopeptidomic 分析的样品制备

This protocol describes a method developed to identify endogenous peptides in human cerebrospinal fluid (CSF). For this purpose, a previously developed ...

Research

JoVE Journal

Neuroscience

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