Mass spectrometric analysis of cyanogen bromide fragments of integral membrane proteins at the picomole level: application to rhodopsin

Advances in time-of-flight mass spectrometry allow unit mass resolution of proteins and peptides up to about 6000 Da molecular weight. Identification of larger proteins and study of their posttranslational or experimental modifications by mass analysis is greatly enhanced by cleavage into smaller fragments. Most membrane proteins are difficult to mass analyze because of their high hydrophobicity, typical expression in low quantities, and because the detergents commonly used for solubilization may be deleterious to mass analysis. Cleavage with cyanogen bromide is beneficial for analysis of membrane proteins since the methionine cleavage sites are typically located in hydrophobic domains and cleavage at these points reduces the size of the hydrophobic fragments. Cyanogen bromide also gives high cleavage yields and introduces only volatile contaminants. Even after cleavage membrane proteins often contain fragments that are difficult to chromatograph. Matrix-assisted laser desorption ionization mass spectrometry (MALDI MS) is capable of analyzing complex mixtures without chromatography. We present a MALDI MS method that quickly and reliably identifies the cyanogen bromide fragments and posttranslational modifications of reduced and alkylated bovine rhodopsin from as little as 30 pmol of rhodopsin in detergent-solubilized retinal rod disk membranes, using 1-5 pmol of digest per sample. The amino acid sequences of some of the peptides in the digest were confirmed by post source decomposition MS analysis of the same samples. The method appears to be general and applicable to the analysis of membrane proteins and the protein composition of membrane preparations.     

Mass spectrometric analysis of integral membrane proteins: application to complete mapping of bacteriorhodopsins and rhodopsin.

Integral membrane proteins have not been readily amenable to the general methods developed for mass spectrometric (or internal Edman degradation) analysis of soluble proteins. We present here a sample preparation method and high performance liquid chromatography (HPLC) separation system which permits online HPLC-electrospray ionization mass spectrometry (ESI-MS) and -tandem mass spectrometry (MS/MS) analysis of cyanogen bromide cleavage fragments of integral membrane proteins. This method has been applied to wild type (WT) bacteriorhodopsin (bR), cysteine containing mutants of bR, and the prototypical G-protein coupled receptor, rhodopsin (Rh). In the described method, the protein is reduced and the cysteine residues pyridylethylated prior to separating the protein from the membrane. Following delipidation, the pyridylethylated protein is cleaved with cyanogen bromide. The cleavage fragments are separated by reversed phase HPLC using an isopropanol/acetonitrile/aqueous TFA solvent system and the effluent peptides analyzed online with a Finnigan LCQ Ion Trap Mass Spectrometer. With the exception of single amino acid fragments and the glycosylated fragment of Rh, which is observable by matrix assisted laser desorption ionization (MALDI)-MS, this system permits analysis of the entire protein in a single HPLC run. This methodology will enable pursuit of chemical modification and crosslinking studies designed to probe the three dimensional structures and functional conformational changes in these proteins. The approach should also be generally applicable to analysis of other integral membrane proteins.     

Mass spectrometric analysis of the kinetics of in vivo rhodopsin phosphorylation

On stimulation, rhodopsin, the light-sensing protein in the rod cells of the retina, is phosphorylated at several sites on its C terminus as the first step in deactivation. We have developed a mass spectrometry-based method to quantify the kinetics of phosphorylation at each site in vivo. After exposing either a freshly dissected mouse retina or the eye of an anesthetized mouse to a flash of light, phosphorylation and dephosphorylation reactions are terminated by rapidly homogenizing the retina or enucleated eye in 8 M urea. The C-terminal peptide containing all known phosphorylation sites is cleaved from rhodopsin, partially purified by ultracentrifugation, and analyzed by liquid chromatography coupled with mass spectrometry (LCMS). The mass spectrometer responds linearly to the peptide from 10 fmole to 100 pmole. The relative sensitivity to peptides with zero to five phosphates was determined using purified phosphopeptide standards. High pressure liquid chromatography (HPLC) coupled with tandem mass spectrometry (LCMS/MS) was used to distinguish the three primary sites of phosphorylation, Ser 334, Ser 338, and Ser 343. Peptides monophosphorylated on Ser 334 were separable from those monophosphorylated on Ser 338 and Ser 343 by reversed-phase HPLC. Although peptides monophosphorylated at Ser 338 and Ser 343 normally coelute, the relative amounts of each species in the single peak could be determined by monitoring the ratio of specific daughter ions characteristic of each peptide. Doubly phosphorylated rhodopsin peptides with different sites of phosphorylation also were distinguished by LCMS/MS. The sensitivity of these methods was evaluated by using them to measure rhodopsin phosphorylation stimulated either by light flashes or by continuous illumination over a range of intensities.     

Mass spectrometric characterization of membrane integral low molecular weight proteins from photosystem II in barley etioplasts

In Photosystem II (PSII), a high number of plastid encoded and membrane integral low molecular weight proteins smaller than 10 kDa, the proteins PsbE, F, H, I, J, K, L, M, N, Tc, Z and the nuclear encoded PsbW, X, Y1, Y2 proteins have been described. Here we show that all low molecular weight proteins of PSII already accumulate in the etioplast membrane fraction in darkness, whereas PsaI and PsaJ of photosystem I (PSI) represent the only low molecular weight proteins that do not accumulate in darkness. We found by BN‐PAGE separation of membrane protein complexes and selective MS that the accumulation of one‐helix proteins from PSII is light independent and occurs in etioplasts. In contrast, in chloroplasts isolated from light‐grown plants, low molecular weight proteins were found to specifically accumulate in PSI and II complexes. Our results demonstrate how plants grown in darkness prepare for the induction of chlorophyll dependent photosystem assembly upon light perception. We anticipate that our investigation will provide the essential means for the analysis of protein assembly in any membrane utilizing low molecular weight protein subunits.     

Mass spectrometric analysis of lysine ubiquitylation reveals promiscuity at site level

The covalent attachment of ubiquitin to proteins regulates numerous processes in eukaryotic cells. Here we report the identification of 753 unique lysine ubiquitylation sites on 471 proteins using higher-energy collisional dissociation on the LTQ Orbitrap Velos. In total 5756 putative ubiquitin substrates were identified. Lysine residues targeted by the ubiquitin-ligase system show no unique sequence feature. Surface accessible lysine residues located in ordered secondary regions, surrounded by smaller and positively charged amino acids are preferred sites of ubiquitylation. Lysine ubiquitylation shows promiscuity at the site level, as evidenced by low evolutionary conservation of ubiquitylation sites across eukaryotic species. Among lysine modifications a significant overlap (20%) between ubiquitylation and acetylation at site level highlights extensive competitive crosstalk among these modifications. This site-specific crosstalk is not prevalent among cell cycle ubiquitylations. Between SUMOylation and ubiquitylation the preferred interaction is through mixed-chain conjugation. Overall these data provide novel insights into the site-specific selection and regulatory function of lysine ubiquitylation.     

Mass spectrometric analysis of glycosylated viral proteins

Introduction: Viral diseases contribute much to human and animal suffering and enormous efforts are directed at developing appropriate vaccines for protection. Glycoproteins constitute much of the viral surfaces and are obvious targets for such vaccine development. This review describes mass spectrometric methods used for the structural determination of these compounds.

Areas covered: The review describes the structures of the N- and O-linked glycans found on glycoproteins and mass spectrometric methods for their ionization and fragmentation. The steps, such as determination of glycan attachment sites and the structures of the attached glycans following their release from the glycoproteins are described and examples are given of the uses of the various analytical methods using mainly influenza, Ebola and HIV as representative examples. Also included are tables listing work on many other viruses.

Expert commentary: Recent technological advances, such as the introduction of ion mobility techniques, have greatly improved analyses in this area and have enabled larger amounts of information to be gathered in shorter time periods on ever smaller amounts of material. Such techniques should greatly accelerate the discovery of vaccine targets and lead to the production of vaccines for diseases not currently available.     

Mass spectrometric characterization of proteins transferred from polyacrylamide gels to membrane filters

In the 1990s, a technique was developed to transfer proteins from electrophoresis gels onto poly(vinylidene difluoride) (PVDF) membranes, digest the proteins on the membranes with proteases such as trypsin and analyze the resulting peptides on the membranes directly by mass spectrometry to identify the proteins. This technique, based on gel electrophoresis, is particularly useful for analyzing protein isoforms, splicing variants and post-translationally modified proteins. Previously, the low ionization efficiency of peptides immobilized on the membranes often rendered this technique useless. However, this technique has been improved by the use of PVDF membranes with a small pore size, which has enabled highly efficient and effective electroblotting and mass spectrometric analyses. Here, the advantage of this technique is discussed.     

Two-dimensional electrophoresis for the isolation of integral membrane proteins and mass spectrometric identification

Acrylamide concentration, urea content, and the trailing ion used for sodium dodecyl sulfate (SDS)-gels modify electrophoretic protein mobilities in a protein-dependent way. Varying these parameters we coupled two SDS-gels to a two-dimensional (2-D) electrophoresis system. Protein spots in 2-D gels are dispersed around a diagonal. Hydrophobic proteins are well separated from water-soluble proteins which is the essential advantage of the novel technique. Mass spectrometric identification of previously unaccessible hydrophobic proteins is now possible.     

Proteolysis and mass spectrometric analysis of an integral membrane: aquaporin 0

Due to hydrophobicity, structural analysis of integral membrane proteins poses a formidable challenge for current mass spectrometry-based proteomics approaches. Herein, we demonstrate results from optimized sample preparation and enzymatic proteolysis procedures for the complete primary structure determination of a targeted integral membrane protein, lens aquaporin 0 (AQP0). Plasma membrane from bovine lens tissue was alkali treated and tryptic digestion was performed in optimized acetonitrile-ammonium bicarbonate solution. Full sequence coverage of AQP0 was observed as tryptic peptides using both matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) and capillary liquid chromatography tandem mass spectrometry (cLC/MS/MS). An amino acid mutation of Thr to Ile/Leu at residue 199 was deduced based on MS/MS results. In a complementary effort to fully sequence the protein, peptic digestion was developed to take advantage of hydrophobic protein solubility in organic acid as well as the decreased activity of pepsin at low pH. Peptic digestion in 10% formic acid (pH 1.2) generated peptides of 500 to 3000 Da and gave 100% sequence coverage by cLC/MS/MS. In addition to post-translational modifications reported previously, a new phosphorylation site at serine 229 and two oxidation sites at tryptophan 202 and 205 were detected on the protein. These methodologies provide complementary detergent- and CNBr-free procedures for detailed analysis of this important membrane channel protein and offer promise for analysis of the integral membrane proteome.     

Mass spectrometry of full-length integral membrane proteins to define functionally relevant structural features

The crystallization and structure determination of integral membrane proteins remains a difficult task relying on a good understanding of the behavior of the protein for success. To date, membrane protein structures are still far outnumbered by soluble protein structures. Mass spectrometry is a powerful and versatile tool offering deep insights into the state of the integral membrane protein the structuralist intends to crystallize. With appropriate sample preparation methods, it provides information that can sometimes prove critical at various stages of the structure determination process, from protein expression to model building. Moreover, valuable knowledge is gained when the identified structural features underlie important functional aspects. Electrospray and matrix assisted laser desorption ionization (MALDI) methods, however, face a particular challenge when dealing with integral membrane proteins. A MALDI method specifically optimized for membrane protein analysis is presented here, with detailed information on the sample preparation and deposition, as well as guidelines for domain determination by limited proteolysis. MALDI-time of flight mass spectrometry can be used to do a proper inventory of initiation sites, to tailor a protein to a stable, well-folded form, and to evaluate selenomethionine replacement. These approaches are illustrated with a few examples drawn from the structural biology of ion channels.     

The topological analysis of integral cytoplasmic membrane proteins

Summary We review three general approaches to determining the topology of integral cytoplasmic membrane proteins. (i) Inspection of the amino acid sequence and use of algorithms to predict membrane spanning segments allows the construction of topological models. For many proteins, the mere identification of such segments and an analysis of the distribution of basic amino acids in hydrophilic domains leads to correct structure predictions. For others, additional factors must come into play in determining topology, (ii) Gene fusion analysis of membrane proteins, in many cases, leads to complete topological models. Such analyses have been carried out in both bacteria and in the yeast Saccharomyces cerevisiae. Conflicts between results from gene fusion analysis and other approaches can be used to explore details of the process of membrane protein assembly. For instance, anomalies in gene fusion studies contributed evidence for the important role of basic amino acids in determining topolog. (iii) Biochemical probes and the site of natural biochemical modifications of membrane proteins give information on their topology. Chemical modifiers, proteases and antibodies made to different domains of a membrane protein can identify which segments of the protein are in the cytoplasm and which are on the extracytoplasmic side of the membrane. Sites of such modifications as glycosylation and phosphorylation help to specify the location of particular hydrophilic domains. The advantages and limitations of these methods are discussed.This work was supported by a fellowship from the National Institute of General Medical Sciences to B.T., by a grant from the National Science Foundation to D.B. and by a grant from the National Institutes of Health to J.B.. J.B. is an American Cancer Society Research Professor.     

Flexible programs for the prediction of average amphipathicity of multiply aligned homologous proteins: application to integral membrane transport proteins

Simple flexible programs (TREEMOMENT and PILEUPMOMENT) are described for depicting the average amphipathicity (hydrophobic moment) along multiply aligned sequences of a family of evolutionarily related proteins. The programs are applicable to any number of aligned sequences and can be set for any desired angle corresponding to a residue repeat unit in a protein secondary structural element such as 100 per residue for an alpha- helix or 180 per residue for a beta-strand. These programs can be used to identify amphipathic regions common to the members of a protein family. The use of these programs is exemplified by showing that some families of integral membrane transport proteins (i.e. permeases of the bacterial phosphotransferase system (PTS) and the anion exchangers of animals) exhibit strikingly amphipathic alpha-helical structures immediately preceding the first hydrophobic transmembrane segment of their membrane-embedded domain(s). Other families, such as the major facilitator superfamily of uniporters, symporters and antiporters, do not exhibit this structural feature. The amphipathic structures in PTS permeases have been implicated in membrane insertion during biogenesis.     

Flexible programs for the prediction of average amphipathicity of multiply aligned homologous proteins: application to integral membrane transport proteins.

Simple flexible programs (TREEMOMENT and PILEUPMOMENT) are described for depicting the average amphipathicity (hydrophobic moment) along multiply aligned sequences of a family of evolutionarily related proteins. The programs are applicable to any number of aligned sequences and can be set for any desired angle corresponding to a residue repeat unit in a protein secondary structural element such as 100 degrees per residue for an alpha-helix or 180 degrees per residue for a beta-strand. These programs can be used to identify amphipathic regions common to the members of a protein family. The use of these programs is exemplified by showing that some families of integral membrane transport proteins (i.e. permeases of the bacterial phosphotransferase system (PTS) and the anion exchangers of animals) exhibit strikingly amphipathic alpha-helical structures immediately preceding the first hydrophobic transmembrane segment of their membrane-embedded domain(s). Other families, such as the major facilitator superfamily of uniporters, symporters and antiporters, do not exhibit this structural feature. The amphipathic structures in PTS permeases have been implicated in membrane insertion during biogenesis.     

Mass spectrometric analysis of cross-linking sites for the structure of proteins and protein complexes

Mass spectrometry has become the method of choice to detect and quantify the minute amounts of proteins at the genomic scale. It has recently been adopted for three dimensional structure analyses of proteins or protein complexes by chemically cross-linking their intact forms and analyzing the cross-linked pieces after digestion. This highlight provides an overview of the technology with a focus on advances in the last two years. This cross-linking mass spectrometry has a great potential to become a powerful tool to supplement current X-ray and NMR method of protein structure analysis.     

Mass spectrometry-from peripheral proteins to membrane motors

That membrane protein complexes could survive in the gas phase had always seemed impossible. The lack of chargeable residues, high hydrophobicity, and poor solubility and the vast excess of detergent contributed to the view that it would not be possible to obtain mass spectra of intact membrane complexes. With the recent success in recording mass spectra of these complexes, first from recombinant sources and later from the cellular environment, many surprising properties of these gas phase membrane complexes have been revealed. The first of these was that the interactions between membrane and soluble subunits could survive in vacuum, without detergent molecules adhering to the complex. The second unexpected feature was that their hydrophobicity and, consequently, lower charge state did not preclude ionization. The final surprising finding was that these gas phase membrane complexes carry with them lipids, bound specifically in subunit interfaces. This provides us with an opportunity to distinguish annular lipids that surround the membrane complexes, from structural lipids that have a role in maintaining structure and subunit interactions. In this perspective, we track these developments and suggest explanations for the various discoveries made during this research.     

Quality control of integral membrane proteins

Integral membrane proteins (IMPs) are essential components of the plasma and organellar membranes of the eukaryotic cell. Non-native IMPs, which can arise as a result of mutations, errors during biosynthesis or cellular stress, can disrupt these membranes and potentially lead to cell death. To protect against this outcome, the cell possesses quality control (QC) systems that detect and dispose of non-native IMPs from cellular membranes. Recent studies suggest that recognition of non-native IMPs by the QC machinery is correlated with the thermodynamic stability of these proteins. Consistent with this, small molecules known as chemical and pharmacological chaperones have been identified that stabilize non-native IMPs and enable them to evade QC. These findings have far-reaching implications for treating human diseases caused by defective IMPs.