Abstract 1130 Capturing membrane snapshots: A quantitative proteome-wide guide for high-throughput spatially-resolved extraction of membrane proteins for structural/functional studies on native membranes

The local membrane environment plays a paramount role in regulating the biology of membrane proteins (MP), including their assembly, functional activation, and degradation. Standard detergent-based MP-solubilization strategies disrupt the native bilayer-environment, leading to a fundamental gap in our understanding of how nanoscale molecular organization of MPs regulates their ability to illicit cellular responses. Addressing this, we have developed a proteome-wide quantitative guide for high-throughput, spatially-resolved extraction of MPs into tunable native nanodiscs of 8-20nm diameter. This enables downstream structural/functional studies of target MPs directly from native membranes. Combining existing MAPs with in-house chain-controlled polymers, we established a library of 15 MAPs. We further developed a high-throughput fluorescence-based assay for rapid screening of synthesized MAPs against target cells to quantitatively evaluate native-nanodisc (MAPdisc) forming capability. Next, we combine MAP extraction with label-free quantitative proteomics to generate a high-throughput proteome-wide quantitative guide that informs the most optimal conditions for spatially-resolved extraction of a target, endogenously expressed MP into MAPdiscs. Applied to HEK293 cells, this culminated in a library of MAP-extraction conditions for >2000 unique MPs. This demonstrates our ability to extract target MPs from their endogenous membranes including plasma membrane along with all intracellular membranes. We have further packaged this information onto a WebApp where users can search for their target MP or multi-MP complex systems to derive the most optimized condition for native extraction into MAPdiscs. We used this library to ascertain optimal solubilization conditions for several multipass MPs, where detergent purification had previously failed. In contrast, our proteomics-guided MAP extraction led to high-purity target MP-containing MAPdiscs. We further couple this rapid MAP-extraction with our recently developed single-molecule Native-Nanobleach platform to demonstrate the power of the approach in high-throughput extraction and subsequent detection of molecular organization of endogenously expressed MPs. The authors acknowledge the following funding sources: NIH R01 GM141192, NIH, RM1 GM149406, ASAP, NSF-GRFP.