Electrospray-ionization mass spectrometry of intact intrinsic membrane proteins.

Membrane proteins drive and mediate many essential cellular processes making them a vital section of the proteome. However, the amphipathic nature of these molecules ensures their detailed structural analysis remains challenging. A versatile procedure for effective electrospray-ionization mass spectrometry (ESI-MS) of intact intrinsic membrane proteins purified using reverse-phase chromatography in aqueous formic acid/isopropanol is presented. The spectra of four examples, bacteriorhodopsin and its apoprotein from Halobacterium and the D1 and D2 reaction-center subunits from spinach thylakoids, achieve mass measurements that are within 0.01% of calculated theoretical values. All of the spectra reveal lesser quantities of other molecular species that can usually be equated with covalently modified subpopulations of these proteins. Our analysis of bovine rhodopsin, the first ESI-MS study of a G-protein coupled receptor, yielded a complex spectrum indicative of extensive molecular heterogeneity. The range of masses measured for the native molecule agrees well with the range calculated based upon variable glycosylation and reveals further heterogeneity arising from other covalent modifications. The technique described represents the most precise way to catalogue membrane proteins and their post-translational modifications. Resolution of the components of protein complexes provides insights into native protein/protein interactions. The apparent retention of structure by bacteriorhodopsin during the analysis raises the potential of obtaining tertiary structure information using more developed ESI-MS experiments.     

“自顶向下(top-down)”的蛋白质组学——蛋白质变体的规模化鉴定

高分辨率质谱技术的快速发展使得"自顶向下"的蛋白质组学(top-down proteomics)研究逐渐成熟起来.在完整蛋白质水平上研究蛋白质组可以提供更精准、更丰富的生物学信息,特别是对于蛋白质上发生了多种关联性的翻译后修饰的情况.另外,由于基因突变、RNA可变剪接和大量蛋白质翻译后修饰的存在,同一个基因往往最终会产生多个"蛋白质变体"(proteoform),而要准确地鉴定这些蛋白质变体,也离不开"自顶向下"的蛋白质组学.在蛋白质水平上的分离技术、质谱技术与生物信息学技术是完整蛋白质鉴定最关键的三项技术.高效的分离技术是实现规模化蛋白质变体鉴定的前提,有效的质谱碎裂是提供可靠鉴定的核心,而快速准确的质谱鉴定算法则是数据分析效率的保障.本文对这三项技术进行了详细总结,重点集中在生物信息学相关技术上,包括对完整蛋白质的质谱数据预处理、数据库搜索鉴定以及翻译后修饰定位等几个计算问题的讨论.     

Integrating 'top-down" and "bottom-up" mass spectrometric approaches for proteomic analysis of Shewanella oneidensis

Here we present a comprehensive method for proteome analysis that integrates both intact protein measurement ("top-down") and proteolytic fragment characterization ("bottom-up") mass spectrometric approaches, capitalizing on the unique capabilities of each method. This integrated approach was applied in a preliminary proteomic analysis of Shewanella oneidensis, a metal-reducing microbe of potential importance to the field of bioremediation. Cellular lysates were examined directly by the "bottom-up" approach as well as fractionated via anion-exchange liquid chromatography for integrated studies. A portion of each fraction was proteolytically digested, with the resulting peptides characterized by on-line liquid chromatography/tandem mass spectrometry. The remaining portion of each fraction containing the intact proteins was examined by high-resolution Fourier transform mass spectrometry. This "top-down" technique provided direct measurement of the molecular masses for the intact proteins and thereby enabled confirmation of post-translational modifications, signal peptides, and gene start sites of proteins detected in the "bottom-up" experiments. A total of 868 proteins from virtually every functional class, including hypotheticals, were identified from this organism.     

Mapping the proteome of thylakoid membranes by de novo sequencing of intermembrane peptide domains

The proteome of a membrane compartment has been investigated by de novo sequence analysis after tryptic in gel digestion. Protein complexes and corresponding protein subunits were separated by a 2-D Blue Native (BN)/SDS-PAGE system. The transmembrane proteins of thylakoid membranes from a higher plant (Hordeum vulgare L.) were identified by the primary sequence of hydrophilic intermembrane peptide domains using nano ESI-MS/MS-analysis. Peptide analysis revealed that lysine residues of membrane proteins are primarily situated in the intermembrane domains. We concluded that esterification of lysine residues with fluorescent dyes may open the opportunity to label membrane proteins still localized in native protein complexes within the membrane phase. We demonstrate that covalent labelling of membrane proteins with the fluorescent dye Cy3 allows high sensitive visualization of protein complexes after 2-D BN/SDS-PAGE. We show that pre-electrophoretic labelling of protein subunits supplements detection of proteins by post-electrophoretic staining with silver and CBB and assists in completing the identification of the membrane proteome.     

Characterization of the 70S Ribosome from Rhodopseudomonas palustris using an integrated "top-down" and "bottom-up" mass spectrometric approach

We present a comprehensive mass spectrometric approach that integrates intact protein molecular mass measurement ("top-down") and proteolytic fragment identification ("bottom-up") to characterize the 70S ribosome from Rhodopseudomonas palustris. Forty-two intact protein identifications were obtained by the top-down approach and 53 out of the 54 orthologs to Escherichia coli ribosomal proteins were identified from bottom-up analysis. This integrated approach simplified the assignment of post-translational modifications by increasing the confidence of identifications, distinguishing between isoforms, and identifying the amino acid positions at which particular post-translational modifications occurred. Our combined mass spectrometry data also allowed us to check and validate the gene annotations for three ribosomal proteins predicted to possess extended C-termini. In particular, we identified a highly repetitive C-terminal "alanine tail" on L25. This type of low complexity sequence, common to eukaryotic proteins, has previously not been reported in prokaryotic proteins. To our knowledge, this is the most comprehensive protein complex analysis to date that integrates two MS techniques.     

Large-scale identification of Caenorhabditis elegans proteins by multidimensional liquid chromatography-tandem mass spectrometry

A proteome of a model organism, Caenorhabditis elegans, was analyzed by an integrated liquid chromatography (LC)-based protein identification system, which was constructed by microscale two-dimensional liquid chromatography (2DLC) coupled with electrospray ionization (ESI) tandem mass spectrometry (MS/MS) on a high-resolution hybrid mass spectrometer with an automated data analysis system. Soluble and insoluble protein fractions were prepared from a mixed growth phase culture of the worm C. elegans, digested with trypsin, and fractionated separately on the 2DLC system. The separated peptides were directly analyzed by on-line ESI-MS/MS in a data-dependent mode, and the resultant spectral data were automatically processed to search a genome sequence database, wormpep 66, for protein identification. The total number of proteins of the composite proteome identified in this method was 1,616, including 110 secreted/targeted proteins and 242 transmembrane proteins. The codon adaptation indices of the identified proteins suggested that the system could identify proteins of relatively low abundance, which are difficult to identify by conventional 2D-gel electrophoresis (GE) followed by an offline mass spectrometric analysis such as peptide mass fingerprinting. Among the approximately 5,400 peptides assigned in this study, many peptides with post-translational modifications, such as N-terminal acetylation and phosphorylation, were detected. This expression profile of C. elegans, containing 571 hypothetical gene products, will serve as the basic data of a major proteome set expressed in the worm.     

Towards functional proteomics of membrane protein complexes: analysis of thylakoid membranes from Chlamydomonas reinhardtii

Functional proteomics of membrane proteins is an important tool for the understanding of protein networks in biological membranes but structural studies on this part of the proteome are limited. In this study we undertook such an approach to analyse photosynthetic thylakoid membranes isolated from wild-type and mutant strains of Chlamydomonas reinhardtii. Thylakoid membrane proteins were separated by high-resolution two-dimensional gel electrophoresis (2-DE) and analysed by immuno-blotting and mass spectrometry for the presence of membrane-spanning proteins. Our data show that light-harvesting complex proteins (LHCP), that cross the membrane with three transmembrane domains, can be separated using this method. We have identified more than 30 different LHCP spots on our gels. Mass spectrometric analysis of 2-DE separated Lhcb1 indicates that this major LHCII protein can associate with the thylakoid membrane with part of its putative transit sequence. Separation of isolated photosystem I (PSI) complexes by 2-DE revealed the presence of 18 LHCI protein spots. The use of two peptide-specific antibodies directed against LHCI subunits supports the interpretation that some of these spots represent products arising from differential processing and post-translational modifications. In addition our data indicate that the reaction centre subunit of PSI, PsaA, that possesses 11 transmembrane domains, can be separated by 2-DE. Comparison between 2-DE maps from thylakoid membrane proteins isolated from a PSI-deficient (Deltaycf4) and a crd1 mutant, which is conditionally reduced in PSI and LHCI under copper-deficiency, showed the presence of most of the LHCI spots in the former but their absence in the latter. Our data demonstrate that (i) hydrophobic membrane proteins like the LHCPs can be faithfully separated by 2-DE, and (ii) that high-resolution 2-DE facilitates the comparative analysis of membrane protein complexes in wild-type and mutants cells.     

Large-scale top-down proteomics of the Arabidopsis thaliana leaf and chloroplast proteomes

We present a large-scale top-down proteomics (TDP) study of plant leaf and chloroplast proteins, achieving the identification of over 4700 unique proteoforms. Using capillary zone electrophoresis coupled with tandem mass spectrometry analysis of offline size-exclusion chromatography fractions, we identify 3198 proteoforms for total leaf and 1836 proteoforms for chloroplast, with 1024 and 363 proteoforms having post-translational modifications, respectively. The electrophoretic mobility prediction of capillary zone electrophoresis allowed us to validate post-translational modifications that impact the charge state such as acetylation and phosphorylation. Identified modifications included Trp (di)oxidation events on six chloroplast proteins that may represent novel targets of singlet oxygen sensing. Furthermore, our TDP data provides direct experimental evidence of the N- and C-terminal residues of numerous mature proteoforms from chloroplast, mitochondria, endoplasmic reticulum, and other sub-cellular localizations. With this information, we suggest true transit peptide cleavage sites and correct sub-cellular localization signal predictions. This large-scale analysis illustrates the power of top-down proteoform identification of post-translational modifications and intact sequences that can benefit our understanding of both the structure and function of hundreds of plant proteins.     

Extraction method for analysis of detergent-solubilized bacteriorhodopsin and hydrophobic peptides by electrospray ionization mass spectrometry

The analysis of integral membrane proteins or transmembrane peptides by electrospray ionization mass spectrometry (ESI-MS) is difficult since detergents, used to solubilize these hydrophobic proteins and peptides, severely suppress analyte ion formation. This problem has been addressed previously by precipitating the protein, removing the detergent, and resolubilizing the protein in a nonpolar solvent. Here, we demonstrate a method that avoids protein precipitation and resolubilization. Detergent-solubilized bacteriorhodopsin is extracted into a nonpolar solvent phase by adding a chloroform/methanol/water solvent mixture to the aqueous detergent solution. ESI mass spectra of the nonpolar, chloroform-rich phase were dominated by peaks due to bacterioopsin. Bacterioopsin precursors with partially cleaved leader sequences were seen in all mass spectra. Additional peaks were likely due to intact bacteriorhodopsin, i.e., bacterioopsin with the retinal prosthetic group attached, and to bacterioopsin associated with lipid molecules. A separation process that occurred in the fused-silica capillary leading to the electrospray tip was essential for obtaining ESI mass spectra of bacterioopsin. The extraction-into-chloroform procedure also worked well with hydrophobic, transmembrane-type peptides that were insoluble in other electrospray solvents, including 100% formic acid, and the method has application to transmembrane peptides formed from digests of integral membrane proteins.     

Integral membrane proteins: bottom-up,top-down and structural proteomics

Introduction: Integral membrane proteins and lipids constitute the bilayer membranes that surround cells and sub-cellular compartments, and modulate movements of molecules and information between them. Since membrane protein drug targets represent a disproportionately large segment of the proteome, technical developments need timely review.

Areas covered: Publically available resources such as Pubmed were surveyed. Bottom-up proteomics analyses now allow efficient extraction and digestion such that membrane protein coverage is essentially complete, making up around one third of the proteome. However, this coverage relies upon hydrophilic loop regions while transmembrane domains are generally poorly covered in peptide-based strategies. Top-down mass spectrometry where the intact membrane protein is fragmented in the gas phase gives good coverage in transmembrane regions, and membrane fractions are yielding to high-throughput top-down proteomics. Exciting progress in native mass spectrometry of membrane protein complexes is providing insights into subunit stoichiometry and lipid binding, and cross-linking strategies are contributing critical in-vivo information.

Expert commentary: It is clear from the literature that integral membrane proteins have yielded to advanced techniques in protein chemistry and mass spectrometry, with applications limited only by the imagination of investigators. Key advances toward translation to the clinic are emphasized.     

The identification and characterization of membranome components

Analyses of proteins from a number of proteomic studies of cell membranes have demonstrated that a significant component of the identified proteins is not predicted to contain transmembrane regions. The presence of such proteins may arise as a result of contamination of the membrane preparations or through real associations. Our aim was to identify integral proteins as well as those that are intimately associated with the microsomal membranes of K562 cells. Isolated membranes were treated under conditions reported to remove noncovalently associated 'peripheral' proteins and the residual proteins were SDS-PAGE-separated and analyzed by LC-MS/MS. Tandem lectin affinity was also examined as a complementary approach for the enrichment of membrane glycoproteins. Approximately 41% of the isolated proteins were assigned as membrane proteins based on the presence of transmembrane regions or covalent post-translational modifications that could account for membrane association. Collectively, these results indicate that there is a significant component of non integral proteins that appear to be as closely associated with membranes as integral elements.     

Post-translational modifications of mitochondrial outer membrane proteins

The mitochondrial outer membrane surrounds the entire organelle. It is composed of a phospholipid bilayer with proteins either embedded into or anchored to the bilayer and mediates the interactions between mitochondria and the rest of the cell. Most of the proteins present in the mitochondrial outer membrane are highly hydrophobic with one or more transmembrane segments. These proteins in conjunction with proteins localized in the inner membrane catalyse energy exchange reactions, the flux of small molecules such as ions, the activation and uptake of long chain fatty acids, import of proteins into the mitochondria, and elimination of biogenic amines among others. In addition, some outer membrane proteins serve as docking sites for non-resident enzymes such as hexokinase and other kinases of signal transduction. All these processes require an intact outer membrane and are highly regulated. One level of regulation with physiological/pathophysiological relevance involves post-translational modification of outer membrane proteins, either by phosphorylation, acetylation or other type of reversible covalent modification. Post-translational modification such as nitration and carbonylation becomes significant under disease states that are associated with increased oxidative stress, i.e. inflammation and ischemia. This review examines the different post-translational modifications of mitochondrial outer membrane proteins and discusses the physiological relevance of these modifications.     

Analysis of intact protein isoforms by mass spectrometry

The diverse proteome of an organism arises from such events as single nucleotide substitutions at the DNA level, different RNA processing, and dynamic enzymatic post-translational modifications. This minireview focuses on the measurement of intact proteins to describe the diversity found in proteomes. The field of biological mass spectrometry has steadily advanced, enabling improvements in the characterization of single proteins to proteins derived from cells or tissues. In this minireview, we discuss the basic technology for "top-down" intact protein analysis. Furthermore, examples of studies involved with the qualitative and quantitative analysis of full-length polypeptides are provided.     

High performance liquid chromatography-electrospray mass spectrometry for the simultaneous resolution and identification of intrinsic thylakoid membrane proteins

In higher plants, both photosystem I (PSI) and II (PSII) consist of membrane-embedded proteins that contain more than one transmembrane alpha helix. PSI is a multiprotein complex consisting of a core complex of thirteen proteins surrounded by four different types of light harvesting antenna proteins. Up to now, the protein components of both photosystems have been characterized by SDS-PAGE and/or immunoblotting and, therefore, identification made only on the basis of electrophoretic mobility, which is sometimes not sufficient to discriminate between individual membrane proteins. This is also complicated by the fact that some proteins, such as the antenna proteins, have almost identical molecular mass and amino acid sequence, making it difficult to identify and ascertain the relative stoichiometry of the proteins. In this paper, we report the complete resolution of the antenna proteins and most of the core components of PSI from spinach, together with the identification of proteins by molecular mass, successfully deduced by the combined use of HPLC coupled on-line with a mass spectrometer equipped with an electrospray ion source (ESI-MS). The proposed RP-HPLC-ESI-MS method holds several advantages over SDS-PAGE, including better protein separation, especially for antenna proteins, mass accuracy, speed, efficiency, and the potential to reveal isomeric forms. Moreover, the molecular masses determined by HPLC-ESI-MS are in good agreement with the molecular masses of the individual components calculated on the basis of their nucleotide-derived amino acid sequences, indicating an absence of post-translational modifications in these proteins. It follows that if the method proposed is useful for these highly hydrophobic proteins, it may be of general use for any membrane proteins, where the presence of detergent for solubilization may compromise their characterization.     

Advances in top-down proteomics for disease biomarker discovery

Top-down mass spectrometry strategies allow identification and characterization of proteins and protein networks by direct fragmentation. These analytical processes involve a panel of fragmentation mechanisms, some of which preserve protein post-translational modifications. Thus top-down is of special interest in clinical biochemistry to probe modified proteins as potential disease biomarkers. This review describes separating methods, mass spectrometry instrumentation, bioinformatics, and theoretical aspects of fragmentation mechanisms used for top-down analysis. The biological interest of this strategy is extensively reported regarding the characterization of post-translational modifications in biochemical pathways and the discovery of biomarkers. One has to bear in mind that quantitative aspects that are beyond the focus of this review are also of critical important for biomarker discovery. The constant evolution of technologies makes top-down strategies crucial players in clinical and basic proteomics.     

Post-translational modifications of mitochondrial outer membrane proteins

Abstract

The mitochondrial outer membrane surrounds the entire organelle. It is composed of a phospholipid bilayer with proteins either embedded into or anchored to the bilayer and mediates the interactions between mitochondria and the rest of the cell. Most of the proteins present in the mitochondrial outer membrane are highly hydrophobic with one or more transmembrane segments. These proteins in conjunction with proteins localized in the inner membrane catalyse energy exchange reactions, the flux of small molecules such as ions, the activation and uptake of long chain fatty acids, import of proteins into the mitochondria, and elimination of biogenic amines among others. In addition, some outer membrane proteins serve as docking sites for non-resident enzymes such as hexokinase and other kinases of signal transduction. All these processes require an intact outer membrane and are highly regulated. One level of regulation with physiological/pathophysiological relevance involves post-translational modification of outer membrane proteins, either by phosphorylation, acetylation or other type of reversible covalent modification. Post-translational modification such as nitration and carbonylation becomes significant under disease states that are associated with increased oxidative stress, i.e. inflammation and ischemia. This review examines the different post-translational modifications of mitochondrial outer membrane proteins and discusses the physiological relevance of these modifications.     

Electron-capture dissociation tandem mass spectrometry

Electron capture dissociation (ECD) is a new fragmentation technique used in Fourier transform ion cyclotron resonance mass spectrometry and is complementary to traditional tandem mass spectrometry techniques. Disulfide bonds, normally stable to vibrational excitation, are preferentially cleaved in ECD. Fragmentation is fast and specific and labile post-translational modifications and non-covalent bonds often remain intact after backbone bond dissociation. ECD provides more extensive sequence coverage in polypeptides, and at higher electron energies even isoleucine and leucine are distinguishable. In biotechnology, the main area of ECD application is expected to be the top-down verification of DNA-predicted protein sequences, de novo sequencing, disulfide bond analysis and the combined top-down/bottom-up analysis of post-translational modifications.