ABSTRACT
Native mass spectrometry (MS) has become an important tool for the analysis of membrane proteins. Although detergent micelles are the most commonly used method for solubilizing membrane proteins for native MS, nanoscale lipoprotein complexes such as nanodiscs are emerging as a promising complementary approach because they solubilize membrane proteins in a lipid bilayer environment. However, prior native MS studies of intact nanodiscs have employed only a limited set of phospholipids that are similar in mass. Here, we extend the range of lipids that are amenable to native MS of nanodiscs by combining lipids with masses that are simple integer multiples of each other. Although these lipid combinations create complex distributions, overlap between resonant peak series allows interpretation of nanodisc spectra containing glycolipids, sterols, and cardiolipin. We also investigate the gas-phase stability of nanodiscs with these new lipids towards collisional activation. We observe that negative ionization mode or charge reduction stabilizes nanodiscs and is essential to preserving labile lipids such as sterols. These new approaches to native MS of nanodiscs will enable future studies of membrane proteins embedded in model membranes that more accurately mimic natural bilayers. Graphical Abstract.
Subject(s)
Lipids/chemistry , Membrane Proteins/analysis , Nanostructures/chemistry , Animals , Cardiolipins/chemistry , Cattle , Cholesterol/chemistry , G(M1) Ganglioside/chemistry , Lipid Bilayers/chemistry , Mass Spectrometry , Models, MolecularABSTRACT
BACKGROUND: Next-generation sequencing techniques such as ChIP-seq allow researchers to investigate the genomic position of nuclear components and events. These experiments provide researchers with thousands of regions of interest to probe in order to identify biological relevance. As the cost of sequencing decreases and its robustness increases, more and more researchers turn to genome-wide studies to better understand the genomic elements they are studying. One way to interpret the output of sequencing studies is to investigate how the element of interest localizes in relationship to genome annotations and the binding of other nuclear components. Colocalization of genomic features could indicate cooperation and provide evidence for more detailed investigations. Although there are several existing tools for visualizing and analyzing colocalization, either they are difficult to use for experimental researchers, not well maintained, or without measurements for colocalization strength. Here we describe an online tool, ColoWeb, designed to allow experimentalists to compare their datasets to existing genomic features in order to generate hypotheses about biological interactions easily and quickly. RESULTS: ColoWeb is a web-based service for evaluating the colocation of genomic features. Users submit genomic regions of interest, for example, a set of locations from a ChIP-seq analysis. ColoWeb compares the submitted regions of interest to the location of other genomic features such as transcription factors and chromatin modifiers. To facilitate comparisons among various genomic features, the output consists of both graphical representations and quantitative measures of the degree of colocalization between user's genomic regions and selected features. Frequent colocation may indicate a biological relationship. CONCLUSION: ColoWeb is a biologist-friendly web service that can quickly provide an assessment of thousands of genomic regions to identify colocated genomic features. ColoWeb is freely available at: http://projects.insilico.us.com/ColoWeb .