Protein ProFiler Microfluidic Electrophoresis System for Proteomics

Innovaregi, Llc, San Jose CA

Investigators

Abstract

Abstract The current estimate of the number of human genes is about 20,700, in contrast to the number of presumed proteins, which likely exceeds one million. This increased complexity is the result of post-transcriptional and post-translational modifications (PTMs) occurring at multiple sites and in a quantitative variable manner. The overwhelming result is a combinatorially expanding complexity of proteoforms that is a challenge to proteomic studies in general and the study of biological mechanisms in particular. Among the new tools utilized in this effort, top-down mass spectrometry (TD MS) analyzes intact proteins with capable mass spectrometers, and in doing so, is ideally suited towards preserving the complexity of multiply modified proteins. However, because the larger intact proteins acquire multiple charged states in the MS, peak capacity is significantly limited. As a consequence, deep mining of the proteome by TD MS is extraordinarily reliant on highly resolving protein separations and quantitative recovery. Most separation systems historically utilized for conventional proteomic mass spectrometry rely on separation systems ideally suited for peptide, not intact proteins. Hence there is high demand for efficient, rapid, and quantitative separations systems, specially designed for intact proteins and TD MS. The lack of such separations technology is a major roadblock to TD MS progress and its wider acceptance. We have developed a separations device that utilizes a specially designed and patented microfluidic glass plate with proprietary coatings that achieves high resolution separations of intact proteins based on truly orthogonal protein properties, size and charge. While similar in principle to conventional 2D gel electrophoresis, our microfluidic Protein ProFiler? avoids rigid gels, MS- incompatible denaturing detergents (e.g., SDS), and accomplishes separations by size, followed by isoelectric focusing to achieve the highest sensitivity. Collection of proteins is accomplished by selective or non-selective means, permitting tailoring the population of collected proteins to the goals of the experiment and MS capabilities. Previously we have demonstrated high performance metrics of the system one dimension at a time. However, this project is focused on completion of the quantitative protein recovery subsystem, encompassed in one specific aim. Successful achievement of the milestone underlying the specific aim and ultimate commercialization will not only demonstrate intact protein separations with quantitative recovery in an automated, MS compatible, liquid-based microfluidic platform, but will play an impactful, enabling role for the TD MS proteomics community, effectively providing the necessary tools to decipher the PTM challenges of modern biology.