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.     

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.     

The thylakoid membrane proteome of two marine diatoms outlines both diatom-specific and species-specific features of the photosynthetic machinery

The thylakoid membrane of photoautotrophic organisms contains the main components of the photosynthetic electron transport chain. Detailed proteome maps of the thylakoid protein complexes of two marine diatoms, Thalassiosira pseudonana and Phaeodactylum tricornutum, were created by means of two-dimensional blue native (BN)/SDS-PAGE coupled with mass spectrometry analysis. One novel diatom-specific photosystem I (PS I)-associated protein was identified. A second plastid-targeted protein with possible PS I interaction was discovered to be restricted to the centric diatom species T. pseudonana. PGR5/PGRL homologues were found to be the only protein components of PS I-mediated cyclic electron transport common to both species. For the first time, evidence for a possible PS I localization of LI818-like light harvesting proteins (Lhcx) is presented. This study also advances the current knowledge on the light harvesting antenna composition and Lhcx expression in T. pseudonana on the protein level and presents details on the molecular distribution of Lhcx in diatoms. Above mentioned proteins and several others with unknown function provide a broad basis for further mutagenesis analysis, aiming toward further understanding of the composition and function of the photosynthetic apparatus of diatoms. The proteomics approach of this study further served as a tool to confirm and improve genome-derived protein models.     

SMA-PAGE: A new method to examine complexes of membrane proteins using SMALP nano-encapsulation and native gel electrophoresis

Most membrane proteins function through interactions with other proteins in the phospholipid bilayer, the cytosol or the extracellular milieu. Understanding the molecular basis of these interactions is key to understanding membrane protein function and dysfunction. Here we demonstrate for the first time how a nano-encapsulation method based on styrene maleic acid lipid particles (SMALPs) can be used in combination with native gel electrophoresis to separate membrane protein complexes in their native state. Using four model proteins, we show that this separation method provides an excellent measure of protein quaternary structure, and that the lipid environment surrounding the protein(s) can be probed using mass spectrometry. We also show that the method is complementary to immunoblotting. Finally we show that intact membrane protein-SMALPs extracted from a band on a gel could be visualised using electron microscopy (EM). Taken together these results provide a novel and elegant method for investigating membrane protein complexes in a native state.     

生物质谱技术在蛋白质组学研究中的应用

随着技术的进步,蛋白质组学的研究重心由最初旨在鉴定细胞或组织内基因组所表达的全部蛋白质转移到从整个蛋白质组水平上阐述包括蛋白翻译后修饰、生物大分子相互作用等反映蛋白质功能的层次。多种质谱离子化技术的突破使质谱技术成为蛋白质组学研究必不可少的手段。质谱技术联合蛋白质组学多角度、深层次探索生命系统分子本质成为现阶段生命科学研究领域的主旋律之一。本文简要综述了肽和蛋白质等生物大分子质谱分析的原理、方式和应用,并对其发展前景做出展望。     

Poxvirus Proteomics and Virus-Host Protein Interactions

Summary: Studies of the functional proteins encoded by the poxvirus genome provide information about the composition of the virus as well as individual virus-virus protein and virus-host protein interactions, which provides insight into viral pathogenesis and drug discovery. Widely used proteomic techniques to identify and characterize specific protein-protein interactions include yeast two-hybrid studies and coimmunoprecipitations. Recently, various mass spectrometry techniques have been employed to identify viral protein components of larger complexes. These methods, combined with structural studies, can provide new information about the putative functions of viral proteins as well as insights into virus-host interaction dynamics. For viral proteins of unknown function, identification of either viral or host binding partners provides clues about their putative function. In this review, we discuss poxvirus proteomics, including the use of proteomic methodologies to identify viral components and virus-host protein interactions. High-throughput global protein expression studies using protein chip technology as well as new methods for validating putative protein-protein interactions are also discussed.     

Protein identification by liquid chromatography-mass spectrometry using retention time prediction

Liquid chromatography has been coupled with mass spectrometry to improve the dynamic range and to reduce the complexity of sample introduced to the mass spectrometer at any given time. The chromatographic separation also provides information on the analytes, such as peptides in enzymatic digests of proteins; information that can be used when identifying the proteins by peptide mass fingerprinting. This paper discusses a recently introduced method based on retention time prediction to extract information from chromatographic separations and the applications of this method to protein identification in organisms with small and large genomes.     

Identification of heme binding protein complexes in murine erythroleukemic cells: study by a novel two-dimensional native separation -- liquid chromatography and electrophoresis

In the current postgenomic era there is a growing interest in analysis of protein complexes in their native state. Here we present a novel two-dimensional separation technique for assessment of native protein complexes. The method combines native chromatography with native electrophoresis. The approach was used to study heme-binding protein complexes in murine erythroleukemia cells. The cells were metabolically labeled with [(59)Fe]-heme and cellular lysates were separated by anion-exchange chromatography. Fractions containing the (59)Fe isotope were collected, concentrated and further separated by native gel electrophoresis. A total of 13 radioactive protein bands were detected and analyzed by liquid chromatography-tandem mass spectrometry. Thirty-three individual proteins were identified and attributed to four novel multiprotein complexes representing four different 'snapshots' of cellular events involved in hemoglobin biosynthesis.     

Quantitative protein stability measurement in vivo

The equilibrium between the native and denatured states of a protein can be key to its function and regulation. Traditionally, the folding equilibrium constant has been measured in vitro using purified protein and simple buffers. However, the biological environment of proteins can differ from these in vitro conditions in ways that could significantly perturb stability. Here, we present the first quantitative comparison between the stability of a protein in vitro and in the cytoplasm of Escherichia coli using amide hydrogen exchange detected by MALDI mass spectrometry (SUPREX). The results indicate that the thermodynamic stability of monomeric lambda repressor within the cell is the same as its stability measured in a simple buffer in vitro. However, when the E. coli are placed in a hyperosmotic environment, the in vivo stability is greatly enhanced. The in vivo SUPREX method provides a general and quantitative way to measure protein stabilities in the cell and will be useful for applications where intracellular stability information provides important biological insights.     

Monitoring macromolecular complexes involved in the chaperonin-assisted protein folding cycle by mass spectrometry

We have used native mass spectrometry to analyze macromolecular complexes involved in the chaperonin-assisted refolding of gp23, the major capsid protein of bacteriophage T4. Adapting the instrumental methods allowed us to monitor all intermediate complexes involved in the chaperonin folding cycle. We found that GroEL can bind up to two unfolded gp23 substrate molecules. Notably, when GroEL is in complex with the cochaperonin gp31, it binds exclusively one gp23. We also demonstrated that the folding and assembly of gp23 into 336-kDa hexamers by GroEL-gp31 can be monitored directly by electrospray ionization mass spectrometry (ESI-MS). These data reinforce the great potential of ESI-MS as a technique to investigate structure-function relationships of protein assemblies in general and the chaperonin-protein folding machinery in particular. A major advantage of native mass spectrometry is that, given sufficient resolution, it allows the analysis at the picomole level of sensitivity of heterogeneous protein complexes with molecular masses up to several million daltons.     

The analysis of protein pharmaceuticals: Near future advances

The analysis of protein pharmaceuticals currently involves a complex series of chromatographic, electrophoretic, spectroscopic, immunological and biological measurements to unequivocally establish their identity, purity and integrity. In this review, I briefly consider the possibility that at least the functional identity and integrity of a protein drug might be established by either a single analysis involving X-ray diffraction, NMR or mass spectrometry, or by a chromatographically based multi-detector system in which a number of critical parameters are essentially simultaneously determined. The use of a protein standard to obtain comparative measurements and new advances in the technology of each of these methods is emphasized. A current major obstacle to the implementation of these approaches is the frequent microheterogeneity of protein preparations. The evolution of biological assays into measurements examining more defined intracellular signal transduction events or based on novel biosensors as well as the analysis of vaccines is also briefly discussed.     

Proteomic Profiling and Neurodegeneration in Alzheimer's Disease

Quantitative proteome analysis of Alzheimer's disease (AD) brains was performed using 2-D gels to identify disease specific changes in protein expression. The task of characterizing the proteome and its components is now practically achievable because of the development and integration of four important tools: protein, EST, and complete genome sequence databases, mass spectrometry, matching software for protein sequences and protein separation technology. Mass spectrometry (MS) instrumentation has undergone a tremendous change over the past decade, culminating in the development of highly sensitive, robust instruments that can reliably analyze biomolecules, particularly proteins and peptides; we identified 35 proteins from over 100 protein spots on a 2-D gel. Using this current technology, protein-expression profiling, which is actually a specialized form of mining, is an important principal application of proteomics. The information obtained has tremendous potential as a means of determining the pathogenesis, and detecting disease markers and potential targets for drug therapy in AD.     

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.     

Intact protein analysis for site-directed mutagenesis overexpression products: plasmid-encoded R67 dihydrofolate reductase

Mass spectrometry is currently the method of choice for the analysis of recombinant protein expression products. By combining proteolytic digestion with peptide mapping and tandem mass spectrometry techniques, verification of site-directed mutagenesis products can be obtained. The proteolytic digestion step converts a purified recombinant protein into a mixture that must be reseparated, thus greatly increasing the analysis time associated with the confirmation of site-directed mutagenesis products. Ion/ion reaction chemistry combined with quadrupole ion trap mass spectrometry provides a fast and efficient way to analyze intact proteins for the correct site-directed mutagenesis products, without heavy reliance on the proteolytic digestion step. Analysis of a series of protein variants (I68M, I68Q, Y69F, and Q67Y) from plasmid-encoded R67 dihydrofolate reductase using ion/ion reaction chemistry confirmed the presence of the correct site-directed mutagenesis products. For the I68M mutant, ion/ion separations detected the presence of extensive degradation from the N-terminal end of the protein. In the case of the Q67Y mutant, a mixture of Q67Y and Q67C species was detected by employing tandem mass spectrometry combined with ion/ion reactions. The ion/ion reaction technique was also performed on a partially purified lysate of the Q67Y/C mixture and successfully screened for the presence of both components in a complex mixture. The ion/ion reaction approach achieved the same results as the proteolytic-digestion-based methodology in a much shorter analysis time.     

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.     

The knowledge-integrated network biomarkers discovery for major adverse cardiac events

The mass spectrometry (MS) technology in clinical proteomics is very promising for discovery of new biomarkers for diseases management. To overcome the obstacles of data noises in MS analysis, we proposed a new approach of knowledge-integrated biomarker discovery using data from Major Adverse Cardiac Events (MACE) patients. We first built up a cardiovascular-related network based on protein information coming from protein annotations in Uniprot, protein-protein interaction (PPI), and signal transduction database. Distinct from the previous machine learning methods in MS data processing, we then used statistical methods to discover biomarkers in cardiovascular-related network. Through the tradeoff between known protein information and data noises in mass spectrometry data, we finally could firmly identify those high-confident biomarkers. Most importantly, aided by protein-protein interaction network, that is, cardiovascular-related network, we proposed a new type of biomarkers, that is, network biomarkers, composed of a set of proteins and the interactions among them. The candidate network biomarkers can classify the two groups of patients more accurately than current single ones without consideration of biological molecular interaction.     

Monitoring enzyme catalysis with mass spectrometry

Mass spectrometry is a rapid, sensitive, and accurate quantitative approach for the direct monitoring of enzyme-catalyzed reactions that does not require a chromophore or radiolabeling and thus provides a viable alternative to existing analytical techniques. In this study the proteolysis of intact viral capsid proteins, the alpha-glucosidase-catalyzed hydrolysis of p-nitrophenyl-alpha-glucopyranoside and the lipoprotein lipase-catalyzed ester hydrolysis of resorufin were examined. Matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry were used to examine the proteolysis of viral protein capsids, providing information about capsid dynamics and the stabilizing force of viral protein/RNA interactions. In addition, k(cat) and K(m) values of enzyme-catalyzed hydrolysis were obtained (without the use of a chromophore). These results also demonstrate the effect an unnatural substrate can have on enzyme activity. Overall, mass spectrometry provides for efficient and quantitative analysis of enzyme-catalyzed reactions, as well as the direct observation of reaction dynamics.     

Isolation and structural characterization of the Ndh complex from mesophyll and bundle sheath chloroplasts of Zea mays

Complex I (NADH: ubiquinone oxidoreductase) is the first complex in the respiratory electron transport chain. Homologs of this complex exist in bacteria, mitochondria and chloroplasts. The minimal complex I from mitochondria and bacteria contains 14 different subunits grouped into three modules: membrane, connecting, and soluble subcomplexes. The complex I homolog (NADH dehydrogenase or Ndh complex) from chloroplasts from higher plants contains genes for two out of three modules: the membrane and connecting subcomplexes. However, there is not much information about the existence of the soluble subcomplex (which is the electron input device in bacterial complex I) in the composition of the Ndh complex. Furthermore, there are contrasting reports regarding the subunit composition of the Ndh complex and its molecular mass. By using blue native (BN)/PAGE and Tricine/PAGE or colorless-native (CN)/PAGE, BN/PAGE and Tricine/PAGE, combined with mass spectrometry, we attempted to obtain more information about the plastidal Ndh complex from maize (Zea mays). Using antibodies, we detected the expression of a new ndh gene (ndhE) in mesophyll (MS) and bundle sheath (BS) chloroplasts and in ethioplasts (ET). We determined the molecular mass of the Ndh complex (550 kDa) and observed that it splits into a 300 kDa membrane subcomplex (containing NdhE) and a 250 kDa subcomplex (containing NdhH, -J and -K). The Ndh complex forms dimers at 1000-1100 kDa in both MS and BS chloroplasts. Native/PAGE of the MS and BS chloroplasts allowed us to determine that the Ndh complex contains at least 14 different subunits. The native gel electrophoresis, western blotting and mass spectrometry allowed us to identify five of the Ndh subunits. We also provide a method that allows the purification of large amounts of Ndh complex for further structural, as well as functional studies.