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

• 批准号: 7788037
• 负责人: JONGYOON HAN
• 金额: $ 23.03万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-01-01 至 2011-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7788037
• 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; inter-alpha-inhibitor; interest; novel; numb protein; operation; pH gradient; promoter; protein complex; public health relevance; tool

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）：鉴于在给定的细胞类型，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通过2D凝胶电泳或质谱法进行后续分析至关重要。具有较高分辨力的等电聚焦（IEF）是用于样品制备的最常见技术之一。 IEF允许基于蛋白质的等电点（PI）的复杂样品分馏。基于PI的分级所需的pH梯度是由载体两极分子或固定的IPG凝胶生成的。在这个项目中，我们提出了一种基于自由流动区电泳的新型连续流，基于无PI的分类技术，该分类技术只能使用常见的缓冲液溶液分割​​蛋白/肽。与其在单个步骤中产生宽的pH梯度并执行平行分级，就像当前的两极分子或凝胶基的IEF技术一样，样品混合物在每个分类步骤后将样品混合物顺序滴定在微流体通道内部，并将其分级为特定的PI范围。顺序排序方案在连续处理模式下起作用，并且可以以1-5 5L/min的流速实现高通量，从而可以在半小时内处理高达100 5L的样品体积。所需的电场是通过强大的自组装珠膜从外部电源提供的。为了使设备制造和操作的简单起见，我们建议在单个芯片中仅对两个分类步骤，并瞄准分子的非常狭窄的PI范围隔离/提取，该范围与D 0.5 pH单元一样窄，来自pH 3-10范围内的复杂样品以及pH范围之外的复杂样品。由于Micromixer中的滴定可以通过滴定自由调节，因此我们可以在酸性中实现狭窄的PI范围隔离，更重要的是，在常规IPG仅提供宽范围条（E PH 3.0单位）的基本范围内。这种连续流分类技术将提供新的可能性，将当前的pH范围扩展到极度酸性（pH <3）或基本条件（pH> 10），这是由于两性和凝胶基于IEF的IEF技术的pH值有限而导致的Terra隐身性。该项目的成功实施将导致一种简单兼容的微流体分类设备，该设备将在极端的PH条件下，在极端的pH条件下，在极端的PH条件下，可以将蛋白质和肽的基于蛋白质和肽的基于PI的样品分馏（D 0.5 pH单位）启用，以适应一个较高的蛋白质，以适应一个目标的蛋白质，即结合RP-LC，它将提供强大的多维分离，用于MS分析。它将为以极端pH值检测蛋白质和肽打开一个新的激动人心的机会。其中一些可能与人类疾病有关，例如胃癌或脑部疾病。因此，该新工具可以改善疾病的检测，并有助于找到在尼比布任务范围内的更好治疗方法。 公共卫生相关性：该项目的目标是开发基于PI的微流体分类设备，该设备允许对复杂蛋白质/肽混合物的连续流和高通量分馏。该设备将能够快速检测和鉴定与某些人类疾病有关的蛋白质。