Nanodiscs are mimics for the study of transmembrane proteins in their surrounding lipid environment. A nanodisc comprises of a lipid bilayer and the membrane scaffold protein (MSP) that acts as the belt in holding the lipid bilayer together. Lipids play an important role in modulating the function of transmembrane proteins. There have been various approaches to determine the lipid composition based on the interactions of lipids and transmembrane proteins in lipid bilayers upon lipid remodeling. Our goal is to use nanodiscs with lipidomics experiments to quantify the lipids in the nanodisc. However, it is challenging to detect both lipids and MSP in the same mass spectrometry experiment, so it is hard to normalize the lipid signal to the total concentration of nanodiscs. Therefore, to quantify the overall lipid composition after lipid remodeling, our objective is to attach a cleavable small molecule conjugate that can serve as an internal standard to track the MSP concentration. MSP was genetically modified to have a cysteine, and the bioconjugate probe will be attached to the MSP via thiol-maleimide chemistry. Upon lipid remodeling and irradiation with UV, the chemical probe attached to the MSP with the photo cleavable linker is expected to release a fragment that would ionize in both positive and negative modes inside the mass spectrometer and be amenable to conventional LC-MS lipidomic workflows. Since the lipids are also ionized at the same time as the ionizable fragment, the amount of each lipid is expected to be calculated based on the peak intensity of the ionizable fragment per a nanodisc.

Design and synthesis of a photo cleavable bioconjugate probe for determining the composition of lipids in a nanodisc

ACS Spring 2022

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