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

描述(申请人提供)：鉴于特定细胞类型、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凝胶电泳法或质谱仪分析是绝对关键的。等电聚焦技术是最常用的样品制备技术之一，具有较高的分辨率。IEF允许根据蛋白质的等电点(PI)对复杂样品进行分级。基于PI的分级所需的pH梯度由载体两性阳离子或固定化IPG凝胶产生。在这个项目中，我们提出了一种新的基于自由流区带电泳的连续流动、无两性阳离子的PI分选技术，该技术只使用一个公共的缓冲液就可以分离复杂的蛋白质/多肽。样品混合物在微流控通道内被顺序滴定，并在每个分选步骤后被分馏到特定的等电点范围，而不是像目前的两性阳离子交换膜或基于凝胶的IEF技术那样，在单一步骤中产生宽的pH梯度并执行平行分馏。顺序分选方案工作在连续处理模式下，可以实现高吞吐量，流速为1-55L/min，允许在半小时内处理高达100 5L的样本量。所需的电场由外部电源通过坚固的自组装珠膜提供。为了简化器件的制作和操作，我们建议在单个芯片中只耦合两个分选步骤，以实现从pH 3-10范围和pH范围以外的复杂样品中分离/提取非常窄的等电点范围的分子，范围窄至d 0.5pH单位。由于等电点范围可通过微型混合器中的滴定自由调整，我们可以在酸性范围内实现窄等电点范围的隔离，更重要的是，在常规IPG仅提供宽范围条带(即pH 3.0单位)的碱性范围内实现等电点范围隔离。这种连续流动分选技术将提供一种新的可能性，将目前的pH范围扩大到极端酸性(pH&lt；3)或碱性条件(pH&gt；10)，由于两性离子交换膜和凝胶基离子交换膜技术的pH能力有限，这是一种隐蔽的陆地。该项目的成功实施将产生一种简单且与下游兼容的微流控分选设备，该设备将能够以高通量模式以高通量模式将蛋白质和多肽以高通量模式自动分级到非常窄的等电点范围(d 0.5pH单位)的蛋白质和多肽，该样品基于等电点，且不含两性离子和凝胶。与RP-LC联用，将为MS分析提供强有力的多维分离。它将为在极端pH值下检测蛋白质和多肽开辟一个新的、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，这一新工具可以改进疾病的检测，并帮助找到更好的治疗方法，这是NIBIB的任务范围。 与公共卫生相关：该项目的目标是开发一种基于等电点的微流控分选设备，允许连续流动和高通量地分离复杂的蛋白质/肽混合物。该设备将使快速检测和鉴定与某些人类疾病有关的蛋白质成为可能。

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.