Development of Microfluidic Electrophoresis Separation Methods for Calmodulin Binding Proteins

Abstract

Calmodulin (CaM) is a Ca2+ signaling protein that regulates more than 100 different enzymes in many intracellular pathways. Investigation of this complex CaM-binding "interactome" requires a sensitive and rapid screening mechanism. The objective of this research work is to develop a highly sensitive fluorescence-based detection method coupled with microfluidic electrophoresis separation assay for CBPs. A functional microfluidic separation platform with red laser-induced fluorescent detection was developed. It is a semi-automated system with integrated functional modules, a separation module, an optical module, a detection module and a control module. AF647-labeled CaM, BSA and concanavalin A were separated to test the microchip platform. Different microfluidic devices, separation modes, and separation conditions were used to optimize the separation of a mixture of the standard proteins. Additionally, the three standards were separated in 100 s by capillary zone electrophoresis-based methods using glass chips. Si-nanoparticle colloidal array chips provided better resolution and separation efficiency in comparison with the glass chips. A photochemical bi-functional cross-linker was used to make a covalent link between AF647-labeled CaM and CBP to allow separation under denaturing conditions. Two CBPs, calcineurin (CN) and eNOS, were used as model proteins and photo cross-linked with CaM using different photochemical cross-linkers (BPM and NHS-diazirine). Mass spectrometric analysis of the in-gel digested sample revealed the presence of both CaM and CBPs in the sample, meaning that CaM and CBPs were successfully cross-linked. NHS-LC-SDA was used as the photo chemical cross-linker; and CaM-CN and CaM-eNOS photoproducts were separated on a PDMS/glass, PDMS/PMMA, glass and Si-nanoparticle colloidal array microfluidic device. CaMAF647 and the individual photoproducts were identified by different separation devices and modes. Overall, this work demonstrated the separation of CaM binding model proteins using different microfluidic devices operated under electrophoresis.