Nanodiscs can preferentially enrich transmembrane proteins in a bottom-up proteomic analysis workflow
Abstract
Membrane proteins are critically important pharmaceutical targets and thus their analytical characterization is vital to drug development efforts. However, compared to cytosolic proteins, membrane proteins are generally more difficult to purify and/or analytically characterize in part due to their amphipathic nature. Nanodiscs (NDs) have become a popular membrane mimetic system offering a well-defined lipid bilayer environment to stabilize membrane proteins for in vitro analyses. NDs based on membrane scaffold proteins (MSPs) offer advantages in terms of their amenability to purification and control over lipid composition. Here we investigated the utility of MSP NDs having different diameters and lipid compositions to enrich membrane proteins isolated from cells for bottom-up proteomic characterization. Specifically, we coupled a robust ND formation and purification workflow with liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis to determine the identity and relative abundance of proteins incorporated into NDs made using different synthesis conditions. We found that the ND diameter had an effect on the relative enrichment of detected proteins and protein classes; however, lipid composition did not have a significant effect. The data indicated that this ND-based approach selectively allowed for the detection of membrane proteins and membrane associated proteins over cytosolic protein contaminants. Furthermore, we found that transmembrane proteins, which are particularly hard to extract and analyze using traditional membrane mimetics, were significantly enriched when using NDs compared to detergent micelles. These results suggest that MSP ND-based approaches could be applied to the selective enrichment of membrane proteins from cells in a manner that could be enabling for both proteomic and other analytical characterization approaches.
Related
- Production of membrane-embedded Bcl-2 proteins - Use of cell-free synthesis in continuous exchange for co-translational insertion of Bcl-2 proteins in lipid bilayer nanodiscs
- Identification and characterization of two transmembrane proteins required for virulence of Ustilago maydis
- Examining roles of varying synthesis and assembly conditions on the incorporation of complex multi-lipid extracts into membrane scaffold protein nanodiscs
- Triphenylphosphonium compounds preferentially inhibit long-chain fatty acid oxidation in cardiac mitochondria
- Optimal bundling of transmembrane helices using sparse distance constraints.
BibTeX
@misc{zheng2026nanodiscs,
title = {{Nanodiscs can preferentially enrich transmembrane proteins in a bottom-up proteomic analysis workflow}},
author = {Hanyu Zheng and Marina C. Sarcinella and Brandon T. Ruotolo and Ryan C. Bailey},
howpublished = {ChemRxiv},
year = {2026},
month = sep,
doi = {10.26434/chemrxiv.15009308/v1},
url = {https://chemrxiv.org/doi/full/10.26434/chemrxiv.15009308/v1},
note = {CC BY},
}
RIS
TY - UNPB
TI - Nanodiscs can preferentially enrich transmembrane proteins in a bottom-up proteomic analysis workflow
AU - Hanyu Zheng
AU - Marina C. Sarcinella
AU - Brandon T. Ruotolo
AU - Ryan C. Bailey
PY - 2026
DA - 2026/09/22
PB - ChemRxiv
DO - 10.26434/chemrxiv.15009308/v1
UR - https://chemrxiv.org/doi/full/10.26434/chemrxiv.15009308/v1
AB - Membrane proteins are critically important pharmaceutical targets and thus their analytical characterization is vital to drug development efforts. However, compared to cytosolic proteins, membrane proteins are generally more difficult to purify and/or analytically characterize in part due to their amphipathic nature. Nanodiscs (NDs) have become a popular membrane mimetic system offering a well-defined lipid bilayer environment to stabilize membrane proteins for in vitro analyses. NDs based on membrane scaffold proteins (MSPs) offer advantages in terms of their amenability to purification and control over lipid composition. Here we investigated the utility of MSP NDs having different diameters and lipid compositions to enrich membrane proteins isolated from cells for bottom-up proteomic characterization. Specifically, we coupled a robust ND formation and purification workflow with liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis to determine the identity and relative abundance of proteins incorporated into NDs made using different synthesis conditions. We found that the ND diameter had an effect on the relative enrichment of detected proteins and protein classes; however, lipid composition did not have a significant effect. The data indicated that this ND-based approach selectively allowed for the detection of membrane proteins and membrane associated proteins over cytosolic protein contaminants. Furthermore, we found that transmembrane proteins, which are particularly hard to extract and analyze using traditional membrane mimetics, were significantly enriched when using NDs compared to detergent micelles. These results suggest that MSP ND-based approaches could be applied to the selective enrichment of membrane proteins from cells in a manner that could be enabling for both proteomic and other analytical characterization approaches.
ER -