Multidimensional Microfluidics for Mapping Post-Translational Modifications

This proposal aims to develop and validate a multidimensional protein separation platform based on polymer microfluidics technology which will be capable of mapping a wide range of post-translational modifications (PTMs) in complex protein samples. The proposed system employs three separation dimensions, namely: IEF, SDS gel electrophoresis, and MALDI-MS. The platform will combine IEF and SDS gel electrophoresis on a single microfluidic chip, with chip-level laser-induced fluorescence detection to record the separated protein positions, and an integrated electrospray tip array to elute separated protein bands from the chip onto a MALDI target plate for MALDI-MS analysis. On-target proteolytic digestion will be performed using two different enzymes to ensure a high degree of peptide coverage during MS analysis. This detection platform will eliminate the manually-intensive process of running standard 2-D gels, while also eliminating the sample dilution associated with traditional 2-D PAGE / MALDI-MS interfacing. Furthermore, the system is expected to provide complete 3-D separations in ~1 hour, approximately an order of magnitude faster than traditional 2-D PAGE / MALDI-MS, with greatly improved PTM analysis capabilities in a miniaturized format.