title: Ultra-thin layer MALDI mass spectrometry of membrane proteins in nanodiscs.
abstract: Nanodiscs have become a leading technology to solubilize membrane proteins for biophysical, enzymatic, and structural investigations. Nanodiscs are nanoscale, discoidal lipid bilayers surrounded by an amphipathic membrane scaffold protein (MSP) belt. A variety of analytical tools has been applied to membrane proteins in nanodiscs, including several recent mass spectrometry studies. Mass spectrometry of full-length proteins is an important technique for analyzing protein modifications, for structural studies, and for identification of proteins present in binding assays. However, traditional matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometry methods for analyzing full-length membrane proteins solubilized in nanodiscs are limited by strong signal from the MSP belt and weak signal from the membrane protein inside the nanodisc. Herein, we show that an optimized ultra-thin layer MALDI sample preparation technique dramatically enhances the membrane protein signal and nearly completely eliminates the MSP signal. First-shot MALDI and MALDI imaging are used to characterize the spots formed by the ultra-thin layer method. Furthermore, the membrane protein enhancement and MSP suppression are shown to be independent of the type of membrane protein and are applicable to mixtures of membrane proteins in nanodiscs.
authors: Department of Chemistry, University of Illinois Urbana-Champaign, Urbana, IL 61801, USA.
authors: Michael T
authors: MT
authors: Marty
authors: Aditi
authors: A
authors: Das
authors: Stephen G
authors: SG
authors: Sligar
mesh_terms: D008051:Lipid Bilayers / Q000737:chemistry*; D008565:Membrane Proteins / Q000032:analysis*; D008567:Membranes, Artificial; D008958:Models, Molecular; D049329:Nanostructures / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
chemical_list: D008051:Lipid Bilayers; D008565:Membrane Proteins; D008567:Membranes, Artificial