Improved method for the analysis of membrane proteins by mass spectrometry.
ABSTRACT: Membrane-bound and membrane-associated proteins are difficult to analyze by mass spectrometry, since the association with lipids impedes the isolation and solubilization of the proteins in buffers suitable for mass spectrometry and the efficient generation of positively charged peptide ions by electrospray ionization. Current methods mostly utilize detergents for the isolation of proteins from membranes. In this study, we present an improved detergent-free method for the isolation and mass spectrometric identification of membrane-bound and membrane-associated proteins. We delipidate proteins from the membrane bilayer by chloroform extraction to overcome dissolution and ionization problems during analysis. Comparison of our results to results obtained by direct tryptic digestion of insoluble membrane pellets identifies an increased number of membrane proteins, and a higher quality of the resulting mass spectral data.
Project description:Vacuoles and lysosomes are single-membrane-bound organelles involved in diverse functions such as intracellular digestion and storage or secretion of metabolites. To understand their origin, evolution and fundamental features, the identification of proteins comprising these compartments in missing links would be invaluable. So, we isolated the vacuoles from Cyanidioschyzon merolae, which is considered to be one of the most primitive photosynthetic eukaryotes, and identified 46 proteins by matrix-assisted laser desorption/ionization time of flight-mass spectrometry. Keywords: peptide mass fingerprinting, MALDI-TOF Overall design: one sample
Project description:Membrane proteins are notoriously challenging to analyze using mass spectrometry (MS) because of their insolubility in aqueous solution. Current MS methods for studying intact membrane proteins involve solubilization in detergent. However, detergents can destabilize proteins, leading to protein unfolding and aggregation, or resulting in inactive entities. Amphipathic polymers, termed amphipols, can be used as a substitute for detergents and have been shown to enhance the stability of membrane proteins. Here, we show the utility of amphipols for investigating the structural and functional properties of membrane proteins using electrospray ionization mass spectrometry (ESI-MS). The functional properties of two bacterial outer-membrane ?-barrel proteins, OmpT and PagP, in complex with the amphipol A8-35 are demonstrated, and their structural integrities are confirmed in the gas phase using ESI-MS coupled with ion mobility spectrometry (IMS). The data illustrate the power of ESI-IMS-MS in separating distinct populations of amphipathic polymers from the amphipol-membrane complex while maintaining a conformationally "nativelike" membrane protein structure in the gas phase. Together, the data indicate the potential importance and utility of amphipols for the analysis of membrane proteins using MS.
Project description:The characterization of membrane proteins is still challenging. The major issue is the high hydrophobicity of membrane proteins that necessitates the use of detergents for their extraction and solubilization. The very poor compatibility of mass spectrometry with detergents remains a tremendous obstacle in studies of membrane proteins. Here, we investigated the potential of atmospheric pressure photoionization (APPI) for mass spectrometry study of membrane proteins. This work was focused on the tetraspanin CD9 and the multidrug transporter BmrA. A set of peptides from CD9, exhibiting a broad range of hydropathicity, was investigated using APPI as compared to electrospray ionization (ESI). Mass spectrometry experiments revealed that the most hydrophobic peptides were hardly ionized by ESI whereas all peptides, including the highly hydrophobic one that corresponds to the full sequence of the first transmembrane domain of CD9, were easily ionized by APPI. The native protein BmrA purified in the presence of the non-ionic detergent beta-D-dodecyl maltoside (DDM) was digested in-solution using trypsin. The resulting peptides were investigated by flow injection analysis of the mixture followed by mass spectrometry. Upon ESI, only detergent ions were detected and the ionic signals from the peptides were totally suppressed. In contrast, APPI allowed many peptides distributed along the sequence of the protein to be detected. Furthermore, the parent ion corresponding to the first transmembrane domain of the protein BmrA was detected under APPI conditions. Careful examination of the APPI mass spectrum revealed a-, b-, c- and y- fragment ions generated by in-source fragmentation. Those fragment ions allowed unambiguous structural characterization of the transmembrane domain. In conclusion, APPI-MS appears as a versatile method allowing the ionization and fragmentation of hydrophobic peptides in the presence of detergent.
Project description:The study of intact soluble protein assemblies by means of mass spectrometry is providing invaluable contributions to structural biology and biochemistry. A recent breakthrough has enabled similar study of membrane protein complexes, following their release from detergent micelles in the gas phase. Careful optimization of mass spectrometry conditions, particularly with respect to energy regimes, is essential for maintaining compact folded states as detergent is removed. However, many of the saccharide detergents widely employed in structural biology can cause unfolding of membrane proteins in the gas phase. Here, we investigate the potential of charge reduction by introducing three membrane protein complexes from saccharide detergents and show how reducing their overall charge enables generation of compact states, as evidenced by ion mobility mass spectrometry. We find that charge reduction stabilizes the oligomeric state and enhances the stability of lipid-bound complexes. This finding is significant since maintaining native-like membrane proteins enables ligand binding to be assessed from a range of detergents that retain solubility while protecting the overall fold.
Project description:Most membrane proteins studies require the use of detergents, but because of the lack of a general, accurate and rapid method to quantify them, many uncertainties remain that hamper proper functional and structural data analyses. To solve this problem, we propose a method based on matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF MS) that allows quantification of pure or mixed detergents in complex with membrane proteins. We validated the method with a wide variety of detergents and membrane proteins. We automated the process, thereby allowing routine quantification for a broad spectrum of usage. As a first illustration, we show how to obtain information of the amount of detergent in complex with a membrane protein, essential for liposome or nanodiscs reconstitutions. Thanks to the method, we also show how to reliably and easily estimate the detergent corona diameter and select the smallest size, critical for favoring protein-protein contacts and triggering/promoting membrane protein crystallization, and to visualize the detergent belt for Cryo-EM studies.
Project description:Membrane protein complexes are commonly introduced to the mass spectrometer solubilized in detergent micelles. The collisional activation used to remove the detergent, however, often causes protein unfolding and dissociation. As in the case for soluble proteins, electrospray in the positive ion mode is most commonly used for the study of membrane proteins. Here we show several distinct advantages of employing the negative ion mode. Negative polarity can yield lower average charge states for membrane proteins solubilized in saccharide detergents, with enhanced peak resolution and reduced adduct formation. Most importantly, we demonstrate that negative ion mode electrospray ionization (ESI) minimizes subunit dissociation in the gas phase, allowing access to biologically relevant oligomeric states. Together, these properties mean that intact membrane protein ions can be generated in a greater range of solubilizing detergents. The formation of negative ions, therefore, greatly expands the possibilities of using mass spectrometry on this intractable class of protein. Graphical Abstract ?.
Project description:BACKGROUND: Detergents are often used to isolate proteins, lipids as well as "detergent-resistant membrane domains" (DRMs) from cells. Different detergents affect different membrane structures according to their physico-chemical properties. However, the effects of different detergents on membrane lysis of boar spermatozoa and the lipid composition of DRMs prepared from the affected sperm membranes have not been investigated so far. RESULTS: Spermatozoa were treated with the selected detergents Pluronic F-127, sodium cholate, CHAPS, Tween 20, Triton X-100 and Brij 96V. Different patterns of membrane disintegration were observed by light and electron microscopy. In accordance with microscopic data, different amounts of lipids and proteins were released from the cells by the different detergents. The biochemical methods to assay the phosphorus and cholesterol contents as well as 31P NMR to determine the phospholipids were not influenced by the presence of detergents since comparable amounts of lipids were detected in the organic extracts from whole cell suspensions after exposure to each detergent. However, matrix-assisted laser desorption and ionization time-of-flight mass spectrometry applied to identify phospholipids was essentially disturbed by the presence of detergents which exerted particular suppression effects on signal intensities. After separation of the membrane fractions released by detergents on a sucrose gradient only Triton X-100 and sodium cholate produced sharp turbid DRM bands. Only membrane solubilisation by Triton X-100 leads to an enrichment of cholesterol, sphingomyelin, phosphatidylinositol and phosphatidylethanolamine in a visible DRM band accompanied by a selective accumulation of proteins. CONCLUSION: The boar sperm membranes are solubilised to a different extent by the used detergents. Particularly, the very unique DRMs isolated after Triton X-100 exposure are interesting candidates for further studies regarding the architecture of sperm.
Project description:Mass spectrometry (MS) applications for intact protein complexes typically require electrospray (ES) ionization and have not been achieved via direct desorption from surfaces. Desorption ES ionization (DESI) MS has however transformed the study of tissue surfaces through release and characterisation of small molecules. Motivated by the desire to screen for ligand binding to intact protein complexes we report the development of a native DESI platform. By establishing conditions that preserve non-covalent interactions we exploit the surface to capture a rapid turnover enzyme-substrate complex and to optimise detergents for membrane protein study. We demonstrate binding of lipids and drugs to membrane proteins deposited on surfaces and selectivity from a mix of related agonists for specific binding to a GPCR. Overall therefore we introduce this native DESI platform with the potential for high-throughput ligand screening of some of the most challenging drug targets including GPCRs.
Project description:Attempts to characterize recombinant integral membrane proteins (IMPs) by mass spectrometry are frequently hindered by several factors including the detergents required for extraction and purification that interferes with analysis, poor solubility, incomplete digestion, and limited identification of the transmembrane domain-spanning peptides. The goal of this study was to examine and develop methods for purification of an IMP that are amenable to downstream digestion of the protein and peptide analysis by mass spectrometry. In this study, we have overexpressed a candidate IMP, the vesicular glutamate transporter 1 (VGLUT1) in Pichia pastoris and examined conditions for the efficient affinity purification, in-solution digestion, and analysis of the protein. Analysis of the intact purified protein without detergent was performed by MALDI-TOF mass spectrometry. The purified IMP was digested with trypsin, and the resulting peptides were identified. A method that utilizes differential solubility and ionization properties of hydrophobic and hydrophilic peptides was developed. Large hydrophobic peptides were only detected in solutions containing 50% formic acid. Ionization of hydrophilic peptides was suppressed in formic acid, but they produced a strong signal in 50% acetonitrile. Eighty-seven percent sequence coverage of the protein was obtained with only one large hydrophobic peptide that remained unidentified. The results demonstrate a simple method to purify and digest a recombinant IMP for analysis by mass spectrometry.
Project description:Native mass spectrometry detection of ligand-protein complexes allowed rapid detection of natural product binders of apo and calcium-bound S100A4 (a member of the metal binding protein S100 family), T cell/transmembrane, immunoglobulin (Ig), and mucin protein 3, and T cell immunoreceptor with Ig and ITIM (immunoreceptor tyrosine-based inhibitory motif) domains precursor protein from extracts and fractions. Based on molecular weight common hits were detected binding to all four proteins. Seven common hits were identified as apigenin 6-C-?-D-glucoside 8-C-?-L-arabinoside, sweroside, 4',5-dihydroxy-7-methoxyflavanone-6-C-rutinoside, loganin acid, 6-C-glucosylnaringenin, biochanin A 7-O-rutinoside and quercetin 3-O-rutinoside. Mass guided isolation and NMR identification of hits confirmed the mass accuracy of the ligand in the ligand-protein MS complexes. Thus, molecular weight ID from ligand-protein complexes by electrospray ionization Fourier transform mass spectrometry allowed rapid dereplication. Native mass spectrometry using electrospray ionization Fourier transform mass spectrometry is a tool for dereplication and metabolomics analysis.