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

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的2D凝胶电泳或质谱分析至关重要。等电聚焦（IEF）以其高分辨能力，是样品制备中最常用的技术之一。IEF允许基于蛋白质的等电点（pI）对复杂样品进行分馏。pi基分馏所需的pH梯度是由载体两性水解物或固定化IPG凝胶产生的。在本项目中，我们提出了一种基于自由流动区带电泳的新型连续流、无两性聚合物的pi分选技术，该技术仅使用通用缓冲溶液就可以分离复杂的蛋白质/肽。不同于当前基于两性电解质或凝胶的IEF技术在单一步骤中产生广泛的pH梯度并进行平行分选，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选到特定的pI范围。顺序分选方案在连续处理模式下工作，可实现高吞吐量，流量为1-5 5L/min，可在半小时内处理高达100 5L的样品量。所需的电场由外部电源通过坚固的自组装头膜提供。为了简化设备制造和操作，我们建议在单个芯片中耦合两个分选步骤，并针对非常窄的pI范围内分离/提取分子，范围窄至d 0.5 pH单位，从pH 3-10范围以及超出pH范围的复杂样品中。由于pI范围可以通过微混合器中的滴定自由调节，因此我们可以在酸性中实现窄pI范围隔离，更重要的是，在常规IPG仅提供宽范围条带（即pH 3.0单位）的基本范围内。这种连续流分选技术将提供一种新的可能性，将当前的pH范围扩展到极酸性（pH < 3）或碱性条件（pH bbb10），这是由于两性电解质和凝胶基IEF技术的pH能力有限而未知的领域。该项目的成功实施将产生一种简单且下游兼容的微流控分选装置，即使在极端pH条件下，也可以在高通量模式下，将蛋白质和肽的自动、无两性电解质和无凝胶、基于pI的样品分离到非常窄的pI范围（d 0.5 pH单位），以适应靶向蛋白质组学的需求。与RP- LC相结合，将为质谱分析提供强大的多维分离。它将为在极端pH值下检测蛋白质和肽开辟一个新的、令人兴奋的机会。其中一些可能与人类疾病有关，如胃癌或脑部疾病。因此，这种新工具可以改进疾病的检测，并有助于找到更好的治疗方法，这是NIBIB的使命范围。

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.