采样使用超探头棒棒糖样的土著人的唾液多肽：简化和增强肽的临床质谱检测
Summary
考虑为今后的临床应用唾液取样，棒棒糖般超滤（LLUF）的探针制作，以适应在人类口腔。直接未消化唾液NanoLC-LTQ质谱分析表明LLUF探头的能力，消除大量的蛋白质和高丰度蛋白，使更多检测低丰度肽。
Abstract
虽然人类唾液蛋白质和多肽已显示1-2 majorly确定从唾液蛋白的胰蛋白酶消化。土著人的唾液多肽的鉴定没有事先与外源酶消化成为当务之急，因为在人类唾液中的原生肽提供的潜在价值，为诊断疾病，预测疾病的进展，并监测治疗效果。适当的采样是提高土著人的唾液多肽鉴定的关键一步。抽样涉及人类唾液离心去除碎片3-4传统方法可能太费时，是适用于临床使用。此外，通过离心去除碎片可能无法清理，大部分受感染的病原体和去除高丰度蛋白质，常常阻碍了鉴定低丰度多肽。
传统的蛋白质组学方法，PRI共轭玛利莲利用二维凝胶电泳（2-DE）凝胶与凝胶消化能找出很多的唾液蛋白质5-6。然而，这种方法一般是不够敏感的检测低丰度的肽/蛋白质。液相色谱 – 质谱（LC-MS法）为基础的蛋白质组学是一种替代，它可以识别2-DE分离，恕不另行蛋白质。虽然这种方法提供了更高的灵敏度，它一般需要事先样品预分馏7预消化和胰蛋白酶，这使得它难以用于临床。
规避由于样品制备质谱的障碍，我们已经开发出一种技术叫做毛细管超滤（CUF）探针8-11。从我们的实验室数据表明，的CUF探测器能够捕捉各种动物在一个充满活力和微创的方式8微环境的蛋白质在体内 –11。没有离心是必要的，因为通过简单的样品采集过程中撤回注射器负压创建。 CUF探针结合的LC-MS已成功确定了胰蛋白酶消化的蛋白质8-11。在这项研究中，我们创建一个棒棒糖般的超滤（LLUF）探头，可以很容易地适合于人类口腔升级的超取样技术。没有胰蛋白酶消化的LC-MS直接分析表明，人类唾液本国包含许多肽来自各种蛋白质的片段。 LLUF探头采样唾液避免离心，但有效地消除了许多大型及高丰度蛋白。质谱结果表明，许多低丰度肽后，筛选出较大的蛋白质与LLUF探针检测。多步样品色谱分离检测低丰度唾液肽独立。为临床应用，LLUF探头纳入D可以用LC-MS在未来可能被用来从唾液，以监测疾病的进展。
Protocol
1。创建LLUF探头聚醚砜膜（2厘米2）密封边界桨环氧树脂胶合膜与的三角形聚丙烯桨（美国加州大学圣迭戈分校）。分子量为30 kDa的截止（截留分子量），聚醚砜与带负电荷的膜。 聚四氟乙烯氟化乙烯丙烯管（内径/外径，0.35/0.50厘米）被连接到了一个三角形的聚丙烯桨缸退出，所以LLUF探头，可以连接到20毫升的注射器。 进入人体的唾液浸泡后，在培养皿中（直径50毫…
Discussion
我们已经发现，许多未消化在人类唾液中存在的多肽片段。这些多肽片段是从富含脯氨酸的蛋白，肌动蛋白，α-淀粉酶，α1球蛋白，β球蛋白，histain 1，1角蛋白，粘蛋白7，聚免疫球蛋白受体，satherin，S100A9的各种形式的衍生工具。有可能是未定的切割位点的多肽生产作出贡献的许多因素。例如，一些多肽片段可能是人类唾液中自然存在的。许多与PQ的C-末端肽（ 表1和补充表1和2）。富…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
这项工作得到了国家卫生资助研究院（以R01-AI067395-01，R21的-R022754-01和R21-I58002-01）。我们感谢尼迈耶C.手稿批判性阅读的。
Materials
| Name of the reagent | Company | Catalog number | Comments |
| Polyethersulfone membranes | Pall Corporation | 30 kDa MWCO | |
| Teflon fluorinated ethylene propylene tube | Upchurch Scientific | ||
| Blue dextran | Sigma | ||
| Nano LC system | Eksigent | ||
| C18 trap column | Agilent | 5065-9913 | |
| LTQ linear ion-trap mass spectrometer | Thermo Fisher | ||
| Sorcerer 2 | Sage-N Research | ||
| Acetonitrile-0.1% formic acid | J.T. Baker | 9832-03 | LC/MS grade |
| Water-0.1% formic acid | J.T. Baker | 9834-03 | LC/MS grade |
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Tags
Human Indigenous Saliva PeptidomeLollipop-like Ultrafiltration ProbePeptide DetectionClinical Mass SpectrometrySaliva ProteomeTryptic DigestsExogenous EnzymesNative PeptidesDisease DiagnosisDisease Progression PredictionTherapeutic Efficacy MonitoringSampling MethodsDebris RemovalInfected PathogensHigh Abundance ProteinsLow Abundance PeptidomeConventional Proteomic ApproachesTwo-dimensional Gel Electrophoresis (2-DE)In-gel DigestionLiquid Chromatography-Mass Spectrometry (LC-MS)
Citazione di questo articolo
Zhu, W., Gallo, R. L., Huang, C. Sampling Human Indigenous Saliva Peptidome Using a Lollipop-Like Ultrafiltration Probe: Simplify and Enhance Peptide Detection for Clinical Mass Spectrometry. J. Vis. Exp. (66), e4108, doi:10.3791/4108 (2012).