Functional genomics by mass spectrometry

Systematic analysis of the function of genes can take place at the oligonucleotide or protein level. The latter has the advantage of being closest to function, since it is proteins that perform most of the reactions necessary for the cell. For most protein based ('proteomic') approaches to gene function, mass spectrometry is the method of choice. Mass spectrometry can now identify proteins with very high sensitivity and medium to high throughput. New instrumentation for the analysis of the proteome has been developed including a MALDI hybrid quadrupole time of flight instrument which combines advantages of the mass finger printing and peptide sequencing methods for protein identification. New approaches include the isotopic labeling of proteins to obtain accurate quantitative data by mass spectrometry, methods to analyze peptides derived from crude protein mixtures and approaches to analyze large numbers of intact proteins by mass spectrometry directly. Examples from this laboratory illustrate biological problem solving by modern mass spectrometric techniques. These include the analysis of the structure and function of the nucleolus and the analysis of signaling complexes.     

A comprehensive set of protein complexes in yeast: mining large scale protein-protein interaction screens

MOTIVATION: The analysis of protein-protein interactions allows for detailed exploration of the cellular machinery. The biochemical purification of protein complexes followed by identification of components by mass spectrometry is currently the method, which delivers the most reliable information--albeit that the data sets are still difficult to interpret. Consolidating individual experiments into protein complexes, especially for high-throughput screens, is complicated by many contaminants, the occurrence of proteins in otherwise dissimilar purifications due to functional re-use and technical limitations in the detection. A non-redundant collection of protein complexes from experimental data would be useful for biological interpretation, but manual assembly is tedious and often inconsistent. RESULTS: Here, we introduce a measure to define similarity within collections of purifications and generate a set of minimally redundant, comprehensive complexes using unsupervised clustering. AVAILABILITY: Programs and results are freely available from http://www.bork.embl-heidelberg.de/Docu/purclust/     

Affinity purification-mass spectrometry. Powerful tools for the characterization of protein complexes.

Multi-protein complexes are emerging as important entities of biological activity inside cells that serve to create functional diversity by contextual combination of gene products and, at the same time, organize the large number of different proteins into functional units. Many a time, when studying protein complexes rather than individual proteins, the biological insight gained has been fundamental, particularly in cases in which proteins with no previous functional annotation could be placed into a functional context derived from their 'molecular environment'. In this minireview, we summarize the current state of the art for the retrieval of multiprotein complexes by affinity purification and their analysis by mass spectrometry. The advances in technology made over the past few years now enable the study of protein complexes on a proteomic scale and it can be anticipated that the knowledge gathered from such projects will fuel drug target discovery and validation pipelines and that the technology is also going to prove valuable in the emerging field of systems biology.     

Large-scale protein identification using mass spectrometry

Recent achievements in genomics have created an infrastructure of biological information. The enormous success of genomics promptly induced a subsequent explosion in proteomics technology, the emerging science for systematic study of proteins in complexes, organelles, and cells. Proteomics is developing powerful technologies to identify proteins, to map proteomes in cells, to quantify the differential expression of proteins under different states, and to study aspects of protein-protein interaction. The dynamic nature of protein expression, protein interactions, and protein modifications requires measurement as a function of time and cellular state. These types of studies require many measurements and thus high throughput protein identification is essential. This review will discuss aspects of mass spectrometry with emphasis on methods and applications for large-scale protein identification, a fundamental tool for proteomics.     

A census of human soluble protein complexes

Cellular processes often depend on stable physical associations between proteins. Despite recent progress, knowledge of the composition of human protein complexes remains limited. To close this gap, we applied an integrative global proteomic profiling approach, based on chromatographic separation of cultured human cell extracts into more than one thousand biochemical fractions that were subsequently analyzed by quantitative tandem mass spectrometry, to systematically identify a network of 13,993 high-confidence physical interactions among 3,006 stably associated soluble human proteins. Most of the 622 putative protein complexes we report are linked to core biological processes and encompass both candidate disease genes and unannotated proteins to inform on mechanism. Strikingly, whereas larger multiprotein assemblies tend to be more extensively annotated and evolutionarily conserved, human protein complexes with five or fewer subunits are far more likely to be functionally unannotated or restricted to vertebrates, suggesting more recent functional innovations.     

Unraveling the dynamics of protein interactions with quantitative mass spectrometry

Knowledge of structure and dynamics of proteins and protein complexes is important to unveil the molecular basis and mechanisms involved in most biological processes. Protein complex dynamics can be defined as the changes in the composition of a protein complex during a cellular process. Protein dynamics can be defined as conformational changes in a protein during enzyme activation, for example, when a protein binds to a ligand or when a protein binds to another protein. Mass spectrometry (MS) combined with affinity purification has become the analytical tool of choice for mapping protein-protein interaction networks and the recent developments in the quantitative proteomics field has made it possible to identify dynamically interacting proteins. Furthermore, hydrogen/deuterium exchange MS is emerging as a powerful technique to study structure and conformational dynamics of proteins or protein assemblies in solution. Methods have been developed and applied for the identification of transient and/or weak dynamic interaction partners and for the analysis of conformational dynamics of proteins or protein complexes. This review is an overview of existing and recent developments in studying the overall dynamics of in vivo protein interaction networks and protein complexes using MS-based methods.     

Probing genuine strong interactions and post-translational modifications in the heterogeneous yeast exosome protein complex

The characterization of heterogeneous multicomponent protein complexes, which goes beyond identification of protein subunits, is a challenging task. Here we describe and apply a comprehensive method that combines a mild affinity purification procedure with a multiplexed mass spectrometry approach for the in-depth characterization of the exosome complex from Saccharomyces cerevisiae expressed at physiologically relevant levels. The exosome is an ensemble of primarily 3' --> 5' exoribonucleases and plays a major role in RNA metabolism. The complex has been reported to consist of 11 proteins in molecular mass ranging from 20 to 120 kDa. By using native macromolecular mass spectrometry we measured accurate masses (around 400 kDa) of several (sub)exosome complexes. Combination of these data with proteolytic peptide LC tandem mass spectrometry using a linear ion trap coupled to a FT-ICR mass spectrometer and intact protein LC mass spectrometry provided us with the identity of the different exosome components and (sub)complexes, including the subunit stoichiometry. We hypothesize that the observed complexes provide information about strongly and weakly interacting exosome-associated proteins. In our analysis we also identified for the first time phosphorylation sites in seven different exosome subunits. The phosphorylation site in the Rrp4 subunit is fully conserved in the human homologue of Rrp4, which is the only previously reported phosphorylation site in any of the human exosome proteins. The described multiplexed mass spectrometry-based procedure is generic and thus applicable to many different types of cellular molecular machineries even if they are expressed at endogenous levels.     

Selective association of protein molecules followed by mass spectrometry.

Nanoflow electrospray mass spectrometry was used to monitor the formation of protein heterodimers of HU proteins from Bacillus stearothermophilus and Bacillus subtilis. This has enabled us to analyze both thermodynamic and kinetic features associated with the dissociation of homodimeric HU proteins. The results obtained correlate well with the kinetics of the protein dissociation process and the free energy difference between homo- and heterodimeric species anticipated from other studies. We suggest that this approach will have general applicability in studying protein association and dissociation under near-equilibrium conditions and will be relevant to a wide range of biological systems.     

Biological insights from hydrogen exchange mass spectrometry

Over the past two decades, hydrogen exchange mass spectrometry (HXMS) has achieved the status of a widespread and routine approach in the structural biology toolbox. The ability of hydrogen exchange to detect a range of protein dynamics coupled with the accessibility of mass spectrometry to mixtures and large complexes at low concentrations result in an unmatched tool for investigating proteins challenging to many other structural techniques. Recent advances in methodology and data analysis are helping HXMS deliver on its potential to uncover the connection between conformation, dynamics and the biological function of proteins and complexes. This review provides a brief overview of the HXMS method and focuses on four recent reports to highlight applications that monitor structure and dynamics of proteins and complexes, track protein folding, and map the thermodynamics and kinetics of protein unfolding at equilibrium. These case studies illustrate typical data, analysis and results for each application and demonstrate a range of biological systems for which the interpretation of HXMS in terms of structure and conformational parameters provides unique insights into function. This article is part of a Special Issue entitled: Mass spectrometry in structural biology.     

Identifying specific protein interaction partners using quantitative mass spectrometry and bead proteomes

The identification of interaction partners in protein complexes is a major goal in cell biology. Here we present a reliable affinity purification strategy to identify specific interactors that combines quantitative SILAC-based mass spectrometry with characterization of common contaminants binding to affinity matrices (bead proteomes). This strategy can be applied to affinity purification of either tagged fusion protein complexes or endogenous protein complexes, illustrated here using the well-characterized SMN complex as a model. GFP is used as the tag of choice because it shows minimal nonspecific binding to mammalian cell proteins, can be quantitatively depleted from cell extracts, and allows the integration of biochemical protein interaction data with in vivo measurements using fluorescence microscopy. Proteins binding nonspecifically to the most commonly used affinity matrices were determined using quantitative mass spectrometry, revealing important differences that affect experimental design. These data provide a specificity filter to distinguish specific protein binding partners in both quantitative and nonquantitative pull-down and immunoprecipitation experiments.     

基于电感耦合等离子体质谱的蛋白质定量技术研究进展

综述了ICP-MS法应用于蛋白质定量技术方面的研究进展.蛋白质定量研究已成为蛋白质组学研究领域的热点,它是解析生物体蛋白质功能的重要途径.基于同位素标记和生物质谱分析技术是蛋白质定量最常用的方法之一,近年来,随着质谱技术的发展,电感耦合等离子体质谱(ICP-MS)技术成为元素测量的重要手段,这使其在蛋白质定量中具一定的应用前景.     

Unraveling the dynamics of protein interactions with quantitative mass spectrometry

Knowledge of structure and dynamics of proteins and protein complexes is important to unveil the molecular basis and mechanisms involved in most biological processes. Protein complex dynamics can be defined as the changes in the composition of a protein complex during a cellular process. Protein dynamics can be defined as conformational changes in a protein during enzyme activation, for example, when a protein binds to a ligand or when a protein binds to another protein. Mass spectrometry (MS) combined with affinity purification has become the analytical tool of choice for mapping protein–protein interaction networks and the recent developments in the quantitative proteomics field has made it possible to identify dynamically interacting proteins. Furthermore, hydrogen/deuterium exchange MS is emerging as a powerful technique to study structure and conformational dynamics of proteins or protein assemblies in solution. Methods have been developed and applied for the identification of transient and/or weak dynamic interaction partners and for the analysis of conformational dynamics of proteins or protein complexes. This review is an overview of existing and recent developments in studying the overall dynamics of in vivo protein interaction networks and protein complexes using MS-based methods.     

Defining the protein complex proteome of plant mitochondria

A classical approach, protein separation by two-dimensional blue native/sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was combined with tandem mass spectrometry and up-to-date computer technology to characterize the mitochondrial "protein complex proteome" of Arabidopsis (Arabidopsis thaliana) in so far unrivaled depth. We further developed the novel GelMap software package to annotate and evaluate two-dimensional blue native/sodium dodecyl sulfate gels. The software allows (1) annotation of proteins according to functional and structural correlations (e.g. subunits of a distinct protein complex), (2) assignment of comprehensive protein identification lists to individual gel spots, and thereby (3) selective display of protein complexes of low abundance. In total, 471 distinct proteins were identified by mass spectrometry, several of which form part of at least 35 different mitochondrial protein complexes. To our knowledge, numerous protein complexes were described for the first time (e.g. complexes including pentatricopeptide repeat proteins involved in nucleic acid metabolism). Discovery of further protein complexes within our data set is open to everybody via the public GelMap portal at www.gelmap.de/arabidopsis_mito.     

Mass spectrometric analysis of protein interactions

Mass spectrometry is a powerful tool for identification of interaction partners and structural characterization of protein interactions because of its high sensitivity, mass accuracy and tolerance towards sample heterogeneity. Several tools that allow studies of protein interaction are now available and recent developments that increase the confidence of studies of protein interaction by mass spectrometry include quantification of affinity-purified proteins by stable isotope labeling and reagents for surface topology studies that can be identified by mass-contributing reporters (e.g. isotope labels, cleavable cross-linkers or fragment ions. The use of mass spectrometers to study protein interactions using deuterium exchange and for analysis of intact protein complexes recently has progressed considerably.     

Quantitative proteome analysis using isotope-coded affinity tags and mass spectrometry

A main objective of proteomics research is to systematically identify and quantify proteins in a given proteome (cells, subcellular fractions, protein complexes, tissues or body fluids). Protein labeling with isotope-coded affinity tags (ICAT) followed by tandem mass spectrometry allows sequence identification and accurate quantification of proteins in complex mixtures, and has been applied to the analysis of global protein expression changes, protein changes in subcellular fractions, components of protein complexes, protein secretion and body fluids. This protocol describes protein-sample labeling with ICAT reagents, chromatographic fractionation of the ICAT-labeled tryptic peptides, and protein identification and quantification using tandem mass spectrometry. The method is suitable for both large-scale analysis of complex samples including whole proteomes and small-scale analysis of subproteomes, and allows quantitative analysis of proteins, including those that are difficult to analyze by gel-based proteomics technology.     

Strategy for identifying protein-protein interactions of gel-separated proteins and complexes by mass spectrometry

A strategy for identifying and characterizing protein interactions among gel-separated proteins and complexes has been developed and tested. The method involves the efficient recovery of proteins or complexes from native gels without affecting their conformational integrity. The use of limited proteolysis of protein complexes, isolated from the gel or formed from the interaction of gel-recovered proteins with potential binding partners, has enabled local binding domains to be efficiently identified using a combination of microfiltration and mass spectrometric analysis. The application of mass spectrometry affords high detection sensitivities, enabling the strategy to be applied to low levels of protein and protein mixtures. The approach is demonstrated for both antigen-antibody and peptide-protein complexes for which protein-binding regions are characterized among simple peptide mixtures and proteolytic digests. The strategy can be easily adapted to achieve high sample throughput and automation using gel-excision robotics and provides a means to study protein interactions in complex biological mixtures and extracts.     

Cross-linking and mass spectrometry methodologies to facilitate structural biology: finding a path through the maze

Multiprotein complexes, rather than individual proteins, make up a large part of the biological macromolecular machinery of a cell. Understanding the structure and organization of these complexes is critical to understanding cellular function. Chemical cross-linking coupled with mass spectrometry is emerging as a complementary technique to traditional structural biology methods and can provide low-resolution structural information for a multitude of purposes, such as distance constraints in computational modeling of protein complexes. In this review, we discuss the experimental considerations for successful application of chemical cross-linking-mass spectrometry in biological studies and highlight three examples of such studies from the recent literature. These examples (as well as many others) illustrate the utility of a chemical cross-linking-mass spectrometry approach in facilitating structural analysis of large and challenging complexes.     

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.     

I-DIRT, a general method for distinguishing between specific and nonspecific protein interactions

Isolation of protein complexes via affinity-tagged proteins provides a powerful tool for studying biological systems, but the technique is often compromised by co-enrichment of nonspecifically interacting proteins. We describe a new technique (I-DIRT) that distinguishes contaminants from bona fide interactors in immunopurifications, overcoming this most challenging problem in defining protein complexes. I-DIRT will be of broad value for studying protein complexes in biological systems that can be metabolically labeled.