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Continuous-flow, Ampholyte-free pI-based Sorting Peptides/proteins at Extreme pH

极端 pH 条件下连续流动、不含两性电解质的基于 pI 的分选肽/蛋白质

基本信息

批准号：
8010211
负责人：
JONGYOON HAN
金额：
$ 18.54万
依托单位：
MASSACHUSETTS INSTITUTE OF TECHNOLOGY
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-01-01 至 2012-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8010211
关键词：
Acute Advanced Development Ampholyte Mixtures Ampholytes Binding Proteins Biological Biological Assay Biological Markers Biology Biomedical Engineering Biosensor Brain Diseases Buffers Cell physiology Cells Charge Clinical Complex Coupling DNA-Binding Proteins Data Detection Devices Disease Disease Marker Electrophoresis Elements Fractionation Gel Genes Goals Histones Hour Immunoassay Ion-Exchange Chromatography Procedure Isoelectric Focusing Isoelectric Point Life Mass Spectrum Analysis Membrane Microfluidic Microchips Microfluidics Mission Myelin National Institute of Biomedical Imaging and Bioengineering Organism Peptides Power Sources Preparation Process Proteins Proteome Proteomics Protocols documentation Reagent Ribosomal Proteins Sampling Scheme Signaling Molecule Solutions Sorting - Cell Movement Source Stomach Carcinoma System Techniques Tissues Titrations Two-Dimensional Gel Electrophoresis Work analytical tool base cell type electric field human disease improved interest novel operation pH gradient pointed protein promoter protein complex public health relevance tool

项目摘要

DESCRIPTION (provided by applicant): In view of the complexity of the expressed proteome in a given cell type, tissue, or organism, proper sample preparation is absolutely critical for subsequent analysis by 2D gel electrophoresis or mass spectrometry. The isoelectric focusing (IEF), with its high resolving power, is one of the most common techniques used for sample preparation. IEF allows a fractionation of a complex sample based on the isoelectric points (pI) of the proteins. The pH gradient necessary for pI-based fractionation is generated either by carrier ampholytes or by immobilized IPG gels. In this project, we propose a novel continuous-flow, ampholyte-free pI-based sorting technique based on free-flow zone electrophoresis that can fractionate complex proteins/peptides only using a common buffer solution. Instead of generating a broad pH gradient and performing a parallel fractionation in a single step, as is the case with the current ampholyte- or gel-based IEF techniques, the sample mixture is sequentially titrated down inside the microfluidic channel and fractionated into a specific pI range after each sorting step. The sequential sorting scheme works in continuous-processing mode and can achieve a high throughput with a flow rate of 1-5 5L/min that allows processing a sample volume up to 100 5L within half an hour. The required electric field is supplied from an external power source through a robust self-assembled bead membrane. For the sake of simplicity in the device fabrication and operation, we propose to couple just two sorting steps in a single chip and aim at a very narrow pI range isolation/extraction of molecules, the range as narrow as d 0.5 pH units, from complex samples in pH 3-10 range as well as beyond pH range. Since the pI range is freely adjustable by the titration in a micromixer, we can achieve a narrow pI range isolation in the acidic and, more importantly, in the basic range where the conventional IPG offers only broad range strips (e pH 3.0 units). This continuous-flow sorting technique will offer a new possibility to extend the current pH range to extremely acidic (pH < 3) or basic conditions (pH > 10), a terra incognita due to the limited pH capability of the ampholyte- and gel-based IEF techniques. A successful implementation of this project will result in a simple and downstream- compatible microfluidic sorting device that will enable an automatic, ampholyte- and gel-free, pI-based sample fractionation of proteins and peptides into a very narrow pI range (d 0.5 pH units), even at extreme pH conditions, in a high-throughput mode to accommodate the needs of targeted proteomics. Combined with RP- LC, it will offer a powerful multidimensional separation for MS analysis. It will open up a new, exciting opportunity to detect proteins and peptides at extreme pH values. Some of them might be related to human diseases such as gastric carcinoma or brain diseases. Thus, this new tool can improve the detection of diseases and help to find better treatment which is within the scope of the mission of NIBIB. PUBLIC HEALTH RELEVANCE: The goal of this project is to develop a microfluidic pI-based sorting device which allows a continuous-flow and high-throughput fractionation of complex protein/peptide mixtures. This device will enable the fast detection and identification of proteins that are related to certain human diseases.

描述（由申请人提供）：鉴于给定细胞类型、组织或生物体中表达的蛋白质组的复杂性，适当的样品制备对于随后通过 2D 凝胶电泳或质谱分析进行分析绝对至关重要。等电聚焦 (IEF) 具有高分辨率，是样品制备中最常用的技术之一。 IEF 允许根据蛋白质的等电点 (pI) 对复杂样品进行分级分离。基于 pI 的分级分离所需的 pH 梯度由载体两性电解质或固定化 IPG 凝胶产生。在这个项目中，我们提出了一种基于自由流动区带电泳的新型连续流动、无两性电解质的基于 pI 的分选技术，该技术可以仅使用常见的缓冲溶液来分级复杂的蛋白质/肽。与当前基于两性电解质或凝胶的 IEF 技术不同，样品混合物在微流体通道内依次滴定，并在每个分选步骤后分级至特定的 pI 范围，而不是在单个步骤中生成广泛的 pH 梯度并进行平行分级。顺序分选方案以连续处理模式工作，可实现1-5 5L/min的高通量，半小时内可处理多达100 5L的样品量。所需的电场由外部电源通过坚固的自组装珠膜提供。为了简化设备制造和操作，我们建议在单个芯片中仅结合两个分选步骤，旨在从 pH 3-10 范围以及超出 pH 范围的复杂样品中以非常窄的 pI 范围分离/提取分子，范围窄至 d 0.5 pH 单位。由于 pI 范围可通过微混合器中的滴定自由调节，因此我们可以在酸性条件下实现窄 pI 范围的分离，更重要的是，在传统 IPG 仅提供宽范围条带（例如 pH 3.0 单位）的碱性范围内。这种连续流分选技术将为将当前 pH 范围扩展到极酸性 (pH < 3) 或碱性条件 (pH > 10) 提供新的可能性，由于基于两性电解质和凝胶的 IEF 技术的 pH 能力有限，这是一个未知领域。该项目的成功实施将产生一种简单且与下游兼容的微流体分选装置，即使在极端 pH 条件下，也能以高通量模式将蛋白质和肽自动、无两性电解质和凝胶、基于 pI 的样品分级分离到非常窄的 pI 范围（d 0.5 pH 单位），以满足目标蛋白质组学的需求。与 RP-LC 相结合，它将为 MS 分析提供强大的多维分离。它将开辟一个新的、令人兴奋的机会来检测极端 pH 值下的蛋白质和肽。其中一些可能与胃癌或脑部疾病等人类疾病有关。因此，这个新工具可以改善疾病的检测并帮助找到更好的治疗方法，这也在 NIBIB 的使命范围内。公共健康相关性：该项目的目标是开发一种基于微流体 pI 的分选装置，该装置允许对复杂的蛋白质/肽混合物进行连续流动和高通量分级分离。该设备将能够快速检测和识别与某些人类疾病相关的蛋白质。