Mapping multiprotein complexes by affinity purification and mass spectrometry

The combination of affinity purification and tandem mass spectrometry (MS) has emerged as a powerful approach to delineate biological processes. In particular, the use of epitope tags has allowed this approach to become scaleable and has bypassed difficulties associated with generation of antibodies. Single epitope tags and tandem affinity purification (TAP) tags have been used to systematically map protein complexes generating protein interaction data at a near proteome-wide scale. Recent developments in the design of tags, optimisation of purification conditions, experimental design and data analysis have greatly improved the sensitivity and specificity of this approach. Concomitant developments in MS, including high accuracy and high-throughput instrumentation together with quantitative MS methods, have facilitated large-scale and comprehensive analysis of multiprotein complexes.     

Delineation of in vivo assembled multiprotein complexes via biomolecular interaction analysis mass spectrometry

Biomolecular interaction analysis mass spectrometry (BIA-MS) is a multiplexed bioanalytical approach used in analysis of proteins from complex biological mixtures. It utilizes surface-immobilized ligands for protein affinity retrieval, surface plasmon resonance for monitoring the ligand-protein interaction and matrix-assisted laser desorption/ionization-time of flight mass spectrometry for revealing the masses of the biomolecules retrieved by the ligand. In order to explore the utility of BIA-MS in delineation of multiprotein complexes, an in vivo assembled protein complex comprised of retinol binding protein (RBP) and transthyretin (TTR) was investigated. Antibodies to RBP and TTR were utilized as ligands in the analysis of the protein complex present in human plasma. The RBP-TTR complex was retrieved by the anti-RBP antibody as indicated by the presence of both RBP and TTR signals in the mass spectra. RBP signals were not observed in the mass spectra of the material retained on the anti-TTR derivatized surface. In addition, the mass-specific detection in BIA-MS allowed detection of RBP and TTR analyte variants.     

Screening transthyretin amyloid fibril inhibitors: characterization of novel multiprotein,multiligand complexes by mass spectrometry

Tetrameric transthyretin is involved in transport of thyroxine and, through its interactions with retinol binding protein, vitamin A. Dissociation of these structures is widely accepted as the first step in the formation of transthyretin amyloid fibrils. Using a mass spectrometric approach, we have examined a series of 18 ligands proposed as inhibitors of this process. The ligands were evaluated for their ability to bind to and stabilize the tetrameric structure, their cooperativity in binding, and their ability to compete with the natural ligand thyroxine. The observation of a novel ten-component complex containing six protein subunits, two vitamin molecules, and two synthetic ligands allows us to conclude that ligand binding does not inhibit association of transthyretin with holo retinol binding protein.     

Structural Insights into retinal guanylylcyclase-GCAP-2 interaction determined by cross-linking and mass spectrometry

The retinal guanylylcyclases ROS-GC 1 and 2 are regulated via the intracellular site by guanylylcyclase-activating proteins (GCAPs). The mechanisms of how GCAPs activate their target proteins remain elusive as exclusively structures of nonactivating calcium-bound GCAP-1 and -2 are available. In this work, we apply a combination of chemical cross-linking with amine-reactive cross-linkers and photoaffinity labeling followed by a mass spectrometric analysis of the created cross-linked products to study the interaction between N-terminally myristoylated GCAP-2 and a peptide derived from the catalytic domain of full-length ROS-GC 1. In our studies, only a few cross-linked products were obtained for calcium-bound GCAP-2, pointing to a well-defined structure of the GCAP-2-GC peptide complex. A much larger number of cross-links were detected in the absence of calcium, indicating a high flexibility of calcium-free GCAP-2 in the complex with the GC peptide. On the basis of the distance constraints imposed by the cross-links, we were able to create a structural model of the calcium-loaded complex between myristoylated GCAP-2 and the GC peptide.     

Cell biology and the genome projects a concerted strategy for characterizing multiprotein complexes by using mass spectrometry

New developments in biological mass spectrometry and the growth of sequence databases are revolutionizing protein analysis. As recent results demonstrate, the high sensitivity and high throughput of this approach allow the rapid characterization of gel-separated proteins and the identification of cognate cDNA and expressed-sequence tag (EST) clones. We advocate here exploiting this new technology for the systematic characterization of multiprotein complexes. The analysis of proteins as components of specific complexes provides an immediate link to biological function and is a powerful method for deciphering the functions of open reading frames uncovered in the genome-sequencing projects.     

Quantitative interactome analysis with chemical cross-linking and mass spectrometry

Structural plasticity and dynamic protein–protein interactions are critical determinants of protein function within living systems. Quantitative chemical cross-linking with mass spectrometry (qXL-MS) is an emerging technology able to provide information on changes in protein conformations and interactions. Importantly, qXL-MS is applicable to complex biological systems, including living cells and tissues, thereby providing insights into proteins within their native environments. Here, we present an overview of recent technological developments and applications involving qXL-MS, including design and synthesis of isotope-labeled cross-linkers, development of new liquid chromatography–MS methodologies, and computational developments enabling interpretation of the data.     

Structural analysis of choline phospholipids by fast atom bombardment mass spectrometry and tandem mass spectrometry

The structures of intact choline phospholipids were determined by positive and negative ion mode fast atom bombardment mass spectrometry, tandem mass spectrometry, and B2/E and B/E constant linked scan mass spectrometry. The molecular weight of the choline lipid could be clearly determined by the appearance of [M + H]+ or [M + Na]+ in the positive ion mode and triplet ions, e.g., [M - 15]-, [M - 60]-, and [M - 86]-, in the negative ion mode. The structures of the triplet ions were assigned to [M - CH3]-, [M - HN(CH3)3]-, and [M - CH2 = CHN(CH3)3]-, respectively, by the MS/MS of each triplet ion, and the origin of the triplet ions was found as the matrix-ion adduct to the target molecule by using the B2/E linked scan technique. The polar group could be identified by the existence of ions indicating glycerophosphocholine and its cleavage products and by the presence of the triplet ions in the negative ion mode. Positional determination of the distribution of constituent fatty acyl groups was carried out by comparing the intensity of deacylated ions from positions 1 and 2 in the positive ion mode and of the ions produced by MS/MS of the triplet ions. From the mass number of the [RCOO]- ion which appeared in the negative ion mode, the molecular weight and degree of unsaturation of the fatty acyl group were determined. The position of double bond(s) in the acyl group was determined from the MS/MS of the [RCOO]- ion.     

Protein complexes and analysis of their assembly by mass spectrometry

The utility of mass spectrometry for the analysis of proteins has grown enormously in the past decade. Significant advances in detection and ionization techniques are allowing questions about noncovalent assembly to be addressed by the direct observation of gas phase complexes, their assembly in real time and their disassembly by perturbation of solution or instrument conditions. These technological innovations have plainly captured the imagination of biological researchers. Recent and novel developments include the combination of mass spectrometry with isotopic labeling, affinity labeling and genomic information. Collectively, these advances are opening new doors to the isolation of complexes, the identification of their substituents and the characterization of their conformations and assembly.     

A mass accuracy sensitive probability based scoring algorithm for database searching of tandem mass spectrometry data

Background  

Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) has become one of the most used tools in mass spectrometry based proteomics. Various algorithms have since been developed to automate the process for modern high-throughput LC-MS/MS experiments.     

Joining forces: integrating proteomics and cross-linking with the mass spectrometry of intact complexes

Protein assemblies are critical for cellular function and understanding their physical organization is the key aim of structural biology. However, applying conventional structural biology approaches is challenging for transient, dynamic, or polydisperse assemblies. There is therefore a growing demand for hybrid technologies that are able to complement classical structural biology methods and thereby broaden our arsenal for the study of these important complexes. Exciting new developments in the field of mass spectrometry and proteomics have added a new dimension to the study of protein-protein interactions and protein complex architecture. In this review, we focus on how complementary mass spectrometry-based techniques can greatly facilitate structural understanding of protein assemblies.     

Protein cross-linking analysis using mass spectrometry, isotope-coded cross-linkers, and integrated computational data processing

Distance constraints in proteins and protein complexes provide invaluable information for calculation of 3D structures, identification of protein binding partners and localization of protein-protein contact sites. We have developed an integrative approach to identify and characterize such sites through the analysis of proteolytic products derived from proteins chemically cross-linked by isotopically coded cross-linkers using LC-MALDI tandem mass spectrometry and computer software. This method is specifically tailored toward the rapid analysis of low microgram amounts of proteins or multimeric protein complexes cross-linked with nonlabeled and deuterium-labeled bis-NHS ester cross-linking reagents (both commercially available and readily synthesized). Through labeling with [18O]water solvent and LC-MALDI analysis, the method further allows the possible distinction between Type 0 and Type 1 or Type 2 modified peptides (monolinks and looplinks or cross-links), although such a distinction is more readily made from analysis of tandem mass spectrometry data. When applied to the bacterial Colicin E7 DNAse/Im7 heterodimeric protein complex, 23 cross-links were identified including six intersubunit cross-links, all between residues that are close in space when examined in the context of the X-ray structure of the heterodimer. In addition, cross-links were successfully identified in five single subunit proteins, beta-lactoglobulin, cytochrome c, lysozyme, myoglobin, and ribonuclease A, establishing the generality of the approach.     

Structural analysis of hydroperoxy- and epoxy-triacylglycerols by liquid chromatography mass spectrometry

Oxidation of triacylglycerols (TAGs) containing oleic acid leads to the formation of several products. This study characterizes hydroperoxy- and epoxy-TAGs including their regio-isomers. For this purpose, epoxy- and hydroperoxy-TAGs, formed by oxidation of 1,2-dipalmitoyl-3-oleoyl-glycerol (PPO) and 1,3-dipalmitoyl-2-oleoyl-glycerol (POP) under air and , were analysed by reverse phase liquid chromatography-electrospray ionisation-mass spectrometry (LC-ESI-MS) using a triple quadrupole mass analyser, in positive ion mode. Post-column infusion of ammonium formiate was used to obtain intense molecular ion adducts. Pure 1,2-dipalmitoyl-3-epoxystearoyl-glycerol (PPEs) and 1,3-dipalmitoyl-2-epoxystearoyl-glycerol (PEsP), synthesized by epoxidation of the corresponding monounsaturated TAGs, were used to confirm MS/MS identification. The use of oxidation experiments permitted unambiguous identification of MS/MS fragmentation pathways of both hydroperoxide and epoxy-TAGs. Fragmentation of hydroperoxy-TAGs are very distinct from their epoxy-TAGs homologues and consist of simultaneous losses of hydrogen peroxide (34 a.m.u.) and water (18 a.m.u.).     

Speeding up tandem mass spectrometry-based database searching by longest common prefix

Background  

Tandem mass spectrometry-based database searching has become an important technology for peptide and protein identification. One of the key challenges in database searching is the remarkable increase in computational demand, brought about by the expansion of protein databases, semi- or non-specific enzymatic digestion, post-translational modifications and other factors. Some software tools choose peptide indexing to accelerate processing. However, peptide indexing requires a large amount of time and space for construction, especially for the non-specific digestion. Additionally, it is not flexible to use.     

Generalized method for probability-based peptide and protein identification from tandem mass spectrometry data and sequence database searching

Tandem mass spectrometry-based proteomics is currently in great demand of computational methods that facilitate the elimination of likely false positives in peptide and protein identification. In the last few years, a number of new peptide identification programs have been described, but scores or other significance measures reported by these programs cannot always be directly translated into an easy to interpret error rate measurement such as the false discovery rate. In this work we used generalized lambda distributions to model frequency distributions of database search scores computed by MASCOT, X!TANDEM with k-score plug-in, OMSSA, and InsPecT. From these distributions, we could successfully estimate p values and false discovery rates with high accuracy. From the set of peptide assignments reported by any of these engines, we also defined a generic protein scoring scheme that enabled accurate estimation of protein-level p values by simulation of random score distributions that was also found to yield good estimates of protein-level false discovery rate. The performance of these methods was evaluated by searching four freely available data sets ranging from 40,000 to 285,000 MS/MS spectra.     

Structural analysis of proteins by laser desorption and electrospray mass spectrometry.

The development of electrospray and matrix-assisted laser desorption mass spectrometry has provided protein chemists with tools for peptide and protein structure analysis with unprecedented sensitivity and molecular weight range. The two technologies can be viewed as competitive with respect to their molecular mass determinations, but complementary with respect to their differences in instrumentation, sample preparation methods, and nature of spectra produced.     

Accurate mass tag retention time database for urine proteome analysis by chromatography-mass spectrometry

Information about peptides and proteins in urine can be used to search for biomarkers of early stages of various diseases. The main technology currently used for identification of peptides and proteins is tandem mass spectrometry, in which peptides are identified by mass spectra of their fragmentation products. However, the presence of the fragmentation stage decreases sensitivity of analysis and increases its duration. We have developed a method for identification of human urinary proteins and peptides. This method based on the accurate mass and time tag (AMT) method does not use tandem mass spectrometry. The database of AMT tags containing more than 1381 AMT tags of peptides has been constructed. The software for database filling with AMT tags, normalizing the chromatograms, database application for identification of proteins and peptides, and their quantitative estimation has been developed. The new procedures for peptide identification by tandem mass spectra and the AMT tag database are proposed. The paper also lists novel proteins that have been identified in human urine for the first time.