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.
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.
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