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 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 is shown to be independent of the type of membrane protein and is applicable to mixtures of membrane proteins in Nanodiscs.

Sample preparation strategies for improving the identification of membrane proteins by mass spectrometry

“…As stated before, both conventional MALDI-TOF and the aforementioned SAMDI assay provided a strong signal from the MSP belt that resulted in a weak signal from the membrane protein inside the nanodisc [65]. To overcome this problem, Marty et al optimized an ultra-thin-layer MALDI sample preparation technique that significantly enhanced the membrane protein signal while almost eliminating the signal from the MSP [65]. The ultra-thinlayer method relied on the preparation of the MALDI sample plate with a thin layer of dried matrix (mainly sinapinic acid) prepared in 1:500:500 TFA/water/acetonitrile.…”

“…This approach allowed one to perform functional assays of membrane-resident proteins, thereby simplifying the procedure and providing molecular information about the analyte. As stated before, both conventional MALDI-TOF and the aforementioned SAMDI assay provided a strong signal from the MSP belt that resulted in a weak signal from the membrane protein inside the nanodisc [65]. To overcome this problem, Marty et al optimized an ultra-thin-layer MALDI sample preparation technique that significantly enhanced the membrane protein signal while almost eliminating the signal from the MSP [65].…”

“…The remaining solution was removed from the plate by aspiration. This method was tested for several proteins and allowed fast and easy analysis of modifications to proteins in nanodiscs; this widened the scope of the technique for structural studies, heterogeneous libraries of membrane proteins in nanodiscs, and novel MSbased assays [65].…”

Nanodiscs as a New Tool to Examine Lipid–Protein Interactions

“…These results demonstrate the effectiveness of approaches combining Nanodisc immunoprecipitations with mass-spectroscopy for clarification of proteins interacting with components in membrane surface. Extensions of this can be widely used for studies of protein–protein, protein–small molecule and protein–lipid interactions (83). …”

Towards integrative structural mass spectrometry: Benefits from hybrid approaches

“…This surprising property, common to other non-ergodic dissociation pathways such as ETD and Surface Induced Dissociation (SID) [68], opened the door to a new area of applications: the mapping of protein-ligand or protein-protein interface by native top-down MS (reviewed in [136]). This approach was applied to different types of ligands including metals [137], nucleotides [138], lipids [139], polyamines [140], and small peptides [135]. At the same time as the development of these top-down approaches, much higher molecular weight proteins were being successfully transmitted in both FT-ICR [6] and Orbitrap [5] instruments.…”