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通过2D凝胶电泳或质谱法进行分析绝对至关重要。等电聚焦（IEF）具有高分辨率，是最常用的样品制备技术之一。IEF允许基于蛋白质的等电点（pI）对复杂样品进行分级。基于pI的分级分离所需的pH梯度通过载体两性电解质或通过固定化IPG凝胶产生。在这个项目中，我们提出了一种新的连续流动，两性电解质免费PI为基础的分选技术的基础上自由流动区带电泳，可以fractionalize复杂的蛋白质/肽只使用一个共同的缓冲溶液。与当前基于两性电解质或凝胶的IEF技术的情况不同，在单个步骤中产生宽pH梯度并进行平行分级，样品混合物在微流体通道内依次向下滴定，并在每个分选步骤后分级至特定的pI范围。顺序分选方案在连续处理模式下工作，并且可以在1-5 5L/min的流速下实现高通量，允许在半小时内处理高达100 5L的样品体积。所需的电场由外部电源通过坚固的自组装珠膜提供。为了简化设备的制造和操作，我们建议在一个单一的芯片耦合只有两个分选步骤，并针对一个非常窄的pI范围的分子分离/提取，范围窄至d 0.5 pH单位，从复杂的样品在pH 3-10的范围，以及超出pH范围。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范围内实现窄pI范围分离，更重要的是，在碱性范围内，传统IPG仅提供宽范围条带（例如pH 3.0单位）。这种连续流分选技术将提供一种新的可能性，将当前的pH范围扩展到极端酸性（pH < 3）或碱性条件（pH > 10），由于两性电解质和基于凝胶的IEF技术的有限pH能力，这是一个未知的领域。该项目的成功实施将产生简单且下游兼容的微流体分选装置，其将能够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的样品分级分离成非常窄的pI范围（d 0.5 pH单位），即使在极端pH条件下，以适应靶向蛋白质组学的需要。与RP-LC联用，将为MS分析提供强大的多维分离.这将为在极端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如胃癌或脑疾病有关。因此，这一新工具可以改善疾病的检测，并有助于找到更好的治疗方法，这属于NIBIB的使命范围。 公共卫生相关性：该项目的目标是开发一种基于PI的微流体分选装置，该装置允许复杂蛋白质/肽混合物的连续流动和高通量分级。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。

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.