Identification of protein-protein interactions using in vivo cross-linking and mass spectrometry

The purification of protein complexes can be accomplished by different types of affinity chromatography. In a typical immunoaffinity experiment, protein complexes are captured from a cell lysate by an immobilized antibody that recognizes an epitope on one of the known components of the complex. After extensive washing to remove unspecifically bound proteins, the complexes are eluted and analyzed by mass spectrometry (MS). Transient complexes, which are characterized by high dissociation constants, are typically lost by this approach. In the present study, we describe a novel method for identifying transient protein-protein interactions using in vivo cross-linking and MS-based protein identification. Live cells are treated with formaldehyde, which rapidly permeates the cell membrane and generates protein-protein cross-links. Proteins cross-linked to a Myc-tagged protein of interest are copurified by immunoaffinity chromatography and subjected to a procedure which dissociates the cross-linked complexes. After separation by SDS-PAGE, proteins are identified by tandem mass spectrometry. Application of this method enabled the identification of numerous proteins that copurified with a constitutively active form of M-Ras (M-Ras(Q71L)). Among these, we identified the RasGAP-related protein IQGAP1 to be a novel interaction partner of M-Ras(Q71L). This method is applicable to many proteins and will aid in the study of protein-protein interactions.     

基于质谱技术的蛋白质互作研究进展

蛋白质作为生命活动的执行者,其功能往往体现在与其他蛋白质的相互作用中,研究蛋白-蛋白相互作用对于人们深入了解和预防传染病、靶向治疗多基因疾病、阐明蛋白质的分子作用机制及各种复杂的生命现象具有重要意义。目前,有多种技术被用来研究蛋白间的相互作用,研究难点在于实时捕获瞬时或弱蛋白质间的相互作用,质谱技术（mass spectrometry, MS）可在某种程度上解决该难点。由于质谱技术可研究简单的蛋白质复合物再到大规模的蛋白质组实验,基于质谱技术研究蛋白质间相互作用被越来越多地应用于科学研究中。综述了蛋白质间相互作用检测方法的研究进展,重点介绍了氢氘交换质谱法和化学交联质谱法研究蛋白质间相互作用的优缺点及其应用,最后对基于质谱技术研究蛋白质间相互作用进行了总结与展望,以期为深入开展相关研究提供借鉴。     

双杂交和质谱技术在蛋白质-蛋白质相互作用研究中的应用

基因的功能是由蛋白质来执行的,而蛋白质要通过与其他生物分子相互作用来完成其各种生物功能。因此,如果能够快速做出蛋白质在不同时间、空间和不同环境中的相互作用图谱,就会帮助我们了解这些蛋白质的功能,进而了解许多生命活动的机制。目前,用于大规模研究蛋白质间相互作用的方法主要有酵母双杂交系统及其衍生系统、亲和纯化与质谱分析联用技术,前者用于研究蛋白分子间的两两相互作用,后者用于研究蛋白质复合物间的相互作用。本文主要阐述了酵母双杂交、细菌双杂交、哺乳动物细胞双杂交、亲和纯化与质谱联用技术在大规模蛋白质相互作用研究中的应用。     

Xlink-identifier: an automated data analysis platform for confident identifications of chemically cross-linked peptides using tandem mass spectrometry

Chemical cross-linking combined with mass spectrometry provides a powerful method for identifying protein-protein interactions and probing the structure of protein complexes. A number of strategies have been reported that take advantage of the high sensitivity and high resolution of modern mass spectrometers. Approaches typically include synthesis of novel cross-linking compounds, and/or isotopic labeling of the cross-linking reagent and/or protein, and label-free methods. We report Xlink-Identifier, a comprehensive data analysis platform that has been developed to support label-free analyses. It can identify interpeptide, intrapeptide, and deadend cross-links as well as underivatized peptides. The software streamlines data preprocessing, peptide scoring, and visualization and provides an overall data analysis strategy for studying protein-protein interactions and protein structure using mass spectrometry. The software has been evaluated using a custom synthesized cross-linking reagent that features an enrichment tag. Xlink-Identifier offers the potential to perform large-scale identifications of protein-protein interactions using tandem mass spectrometry.     

Investigating Protein-protein Interactions in Live Cells Using Bioluminescence Resonance Energy Transfer

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live cells. BRET is the non-radiative transfer of energy from a ''donor'' luciferase enzyme to an ''acceptor'' fluorescent protein. In the most common configuration of this assay, the donor is Renilla reniformis luciferase and the acceptor is Yellow Fluorescent Protein (YFP). Because the efficiency of energy transfer is strongly distance-dependent, observation of the BRET phenomenon requires that the donor and acceptor be in close proximity. To test for an interaction between two proteins of interest in cultured mammalian cells, one protein is expressed as a fusion with luciferase and the second as a fusion with YFP. An interaction between the two proteins of interest may bring the donor and acceptor sufficiently close for energy transfer to occur. Compared to other techniques for investigating protein-protein interactions, the BRET assay is sensitive, requires little hands-on time and few reagents, and is able to detect interactions which are weak, transient, or dependent on the biochemical environment found within a live cell. It is therefore an ideal approach for confirming putative interactions suggested by yeast two-hybrid or mass spectrometry proteomics studies, and in addition it is well-suited for mapping interacting regions, assessing the effect of post-translational modifications on protein-protein interactions, and evaluating the impact of mutations identified in patient DNA.     

Insights into enzyme structure and dynamics elucidated by amide H/D exchange mass spectrometry

Conformational changes and protein dynamics play an important role in the catalytic efficiency of enzymes. Amide H/D exchange mass spectrometry (H/D exchange MS) is emerging as an efficient technique to study the local and global changes in protein structure and dynamics due to ligand binding, protein activation-inactivation by modification, and protein-protein interactions. By monitoring the selective exchange of hydrogen for deuterium along a peptide backbone, this sensitive technique probes protein motions and structural elements that may be relevant to allostery and function. In this report, several applications of H/D exchange MS are presented which demonstrate the unique capability of amide hydrogen/deuterium exchange mass spectrometry for examining dynamic and structural changes associated with enzyme catalysis.     

Amide hydrogen/deuterium exchange & MALDI-TOF mass spectrometry analysis of Pak2 activation

Amide hydrogen/deuterium exchange (H/D exchange) coupled with mass spectrometry has been widely used to analyze the interface of protein-protein interactions, protein conformational changes, protein dynamics and protein-ligand interactions. H/D exchange on the backbone amide positions has been utilized to measure the deuteration rates of the micro-regions in a protein by mass spectrometry(1,2,3). The resolution of this method depends on pepsin digestion of the deuterated protein of interest into peptides that normally range from 3-20 residues. Although the resolution of H/D exchange measured by mass spectrometry is lower than the single residue resolution measured by the Heteronuclear Single Quantum Coherence (HSQC) method of NMR, the mass spectrometry measurement in H/D exchange is not restricted by the size of the protein(4). H/D exchange is carried out in an aqueous solution which maintains protein conformation. We provide a method that utilizes the MALDI-TOF for detection(2), instead of a HPLC/ESI (electrospray ionization)-MS system(5,6). The MALDI-TOF provides accurate mass intensity data for the peptides of the digested protein, in this case protein kinase Pak2 (also called γ-Pak). Proteolysis of Pak 2 is carried out in an offline pepsin digestion. This alternative method, when the user does not have access to a HPLC and pepsin column connected to mass spectrometry, or when the pepsin column on HPLC does not result in an optimal digestion map, for example, the heavily disulfide-bonded secreted Phospholipase A(2;) (sPLA(2;)). Utilizing this method, we successfully monitored changes in the deuteration level during activation of Pak2 by caspase 3 cleavage and autophosphorylation(7,8,9).     

蛋白质复合体及蛋白质相互作用研究新策略—化学交联结合质谱分析法

近年来化学交联法结合质谱分析法被广泛用于蛋白质复合体结构及蛋白质相互作用的研究。研究表明这两种方法的有机结合为研究蛋白质复合体结构及蛋白质相互作用提供了一条新的途径。文章对不同类型的化学交联剂、质谱分析中的Bottom-up 与Top-down 两种研究策略,以及化学交联法结合质谱分析法在蛋白质复合体结构、蛋白质相互作用研究中的应用进行综述。这两种方法的不断发展与完善,将会极大促进生物大分子复合体结构及蛋白质相互作用的研究。     

Affinity mass spectrometry-based approaches for the analysis of protein-protein interaction and complex mixtures of peptide-ligands.

Combined applications of affinity purification procedures and mass-spectrometric analyses (affinity mass spectrometry or affinity-directed mass spectrometry) have gained broad interest in various fields of biological sciences. We have extended these techniques to the purification and analysis of closely related peptides from complex mixtures and to the characterization of binding motifs and relative affinities in protein-protein interactions. The posttranslational modifications in the carboxy-terminal region of porcine brain tubulin are used as an example for the applicability of affinity mass spectrometry in the characterization of complex patterns of related peptides. We also show that affinity mass spectrometry allows the mapping of sequential binding motifs of two interacting proteins. Using the ActA/Mena protein-protein complex as a model system, we show that we can selectively purify Mena-binding peptides from a tryptic digest of ActA. The results from this assay are compared to data sets obtained earlier by classical methods using synthetic peptides and molecular genetic experiments. As a further expansion of affinity mass spectrometry, we have established an internally standardized system that allows comparison of the affinities of related ligands for a given protein. Here the affinities of two peptide ligands for the monoclonal tubulin-specific antibody YL1/2 are determined in terms of half-maximal competition.     

Mass spectrometry-based functional proteomics: from molecular machines to protein networks

The study of protein-protein interactions by mass spectrometry is an increasingly important part of post-genomics strategies to understand protein function. A variety of mass spectrometry-based approaches allow characterization of cellular protein assemblies under near-physiological conditions and subsequent assignment of individual proteins to specific molecular machines, pathways and networks, according to an increasing level of organizational complexity. An appropriate analytical strategy can be individually tailored--from an in-depth analysis of single complexes to a large-scale characterization of entire molecular pathways or even an analysis of the molecular organization of entire expressed proteomes. Here we review different options regarding protein-complex purification strategies, mass spectrometry analysis and bioinformatic methods according to the specific question that is being addressed.     

Predicting direct protein interactions from affinity purification mass spectrometry data

Background  

Affinity purification followed by mass spectrometry identification (AP-MS) is an increasingly popular approach to observe protein-protein interactions (PPI) in vivo. One drawback of AP-MS, however, is that it is prone to detecting indirect interactions mixed with direct physical interactions. Therefore, the ability to distinguish direct interactions from indirect ones is of much interest.     

Extracting gene function from protein-protein interactions using Quantitative BAC InteraCtomics (QUBIC)

Large-scale proteomic screens are increasingly employed for placing genes into specific pathways. Therefore generic methods providing a physiological context for protein-protein interaction studies are of great interest. In recent years many protein-protein interactions have been determined by affinity purification followed by mass spectrometry (AP-MS). Among many different AP-MS approaches, the recently developed Quantitative BAC InteraCtomics (QUBIC) approach is particularly attractive as it uses tagged, full-length baits that are expressed under endogenous control. For QUBIC large cell line collections expressing tagged proteins from BAC transgenes or gene trap loci have been developed and are freely available. Here we describe detailed workflows on how to obtain specific protein binding partners with high confidence under physiological conditions. The methods are based on fast, streamlined and generic purification procedures followed by single run liquid chromatography-mass spectrometric analysis. Quantification is achieved either by the stable isotope labeling of amino acids in cell culture (SILAC) method or by a 'label-free' procedure. In either case data analysis is performed by using the freely available MaxQuant environment. The QUBIC approach enables biologists with access to high resolution mass spectrometry to perform small and large-scale protein interactome mappings.     

PUB-MS: a mass spectrometry-based method to monitor protein-protein proximity in vivo

The common techniques to study protein-protein proximity in vivo are not well adapted to the capabilities and the expertise of a standard proteomics laboratory, typically based on the use of mass spectrometry. With the aim of closing this gap, we have developed PUB-MS (for proximity utilizing biotinylation and mass spectrometry), an approach to monitor protein-protein proximity, based on biotinylation of a protein fused to a biotin-acceptor peptide (BAP) by a biotin-ligase, BirA, fused to its interaction partner. The biotinylation status of the BAP can be further detected by either Western analysis or mass spectrometry. The BAP sequence was redesigned for easy monitoring of the biotinylation status by LC-MS/MS. In several experimental models, we demonstrate that the biotinylation in vivo is specifically enhanced when the BAP- and BirA-fused proteins are in proximity to each other. The advantage of mass spectrometry is demonstrated by using BAPs with different sequences in a single experiment (allowing multiplex analysis) and by the use of stable isotopes. Finally, we show that our methodology can be also used to study a specific subfraction of a protein of interest that was in proximity with another protein at a predefined time before the analysis.     

Protein surface mapping by chemical oxidation: structural analysis by mass spectrometry

The solvent-accessible surface area of proteins is important in biological function for many reasons, including protein-protein interactions, protein folding, and catalytic sites. Here we present a chemical technique to oxidize amino acid side chains in a model protein, apomyoglobin, and subsequent elucidation of the effect of solvent accessibility on the sites of oxidation. Under conditions of low protein oxidation (zero to three oxygen atoms added per apomyoglobin molecule), we have positively identified five oxidation sites by liquid chromatography-tandem mass spectrometry and high-resolution Fourier transform mass spectrometry. Our results indicate that all oxidized amino acids, with the exception of methionine, have highly solvent-accessible side chains, but the rate of oxidation may not be dictated solely by solvent accessibility and amino acid identity.     

ATG7(2) Interacts With Metabolic Proteins and Regulates Central Energy Metabolism

Macroautophagy/autophagy is an essential catabolic process that targets a wide variety of cellular components including proteins, organelles, and pathogens. ATG7, a protein involved in the autophagy process, plays a crucial role in maintaining cellular homeostasis and can contribute to the development of diseases such as cancer. ATG7 initiates autophagy by facilitating the lipidation of the ATG8 proteins in the growing autophagosome membrane. The noncanonical isoform ATG7(2) is unable to perform ATG8 lipidation; however, its cellular regulation and function are unknown. Here, we uncovered a distinct regulation and function of ATG7(2) in contrast with ATG7(1), the canonical isoform. First, affinity-purification mass spectrometry analysis revealed that ATG7(2) establishes direct protein–protein interactions (PPIs) with metabolic proteins, whereas ATG7(1) primarily interacts with autophagy machinery proteins. Furthermore, we identified that ATG7(2) mediates a decrease in metabolic activity, highlighting a novel splice-dependent function of this important autophagy protein. Then, we found a divergent expression pattern of ATG7(1) and ATG7(2) across human tissues. Conclusively, our work uncovers the divergent patterns of expression, protein interactions, and function of ATG7(2) in contrast to ATG7(1). These findings suggest a molecular switch between main catabolic processes through isoform-dependent expression of a key autophagy gene.     

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.     

Proteomics: quantitative and physical mapping of cellular proteins

Genome sequencing provides a wealth of information on predicted gene products (mostly proteins), but the majority of these have no known function. Two-dimensional gel electrophoresis and mass spectrometry have, coupled with searches in protein and EST databases, transformed the protein-identification process. The proteome is the expressed protein complement of a genome and proteomics is functional genomics at the protein level. Proteomics can be divided into expression proteomics, the study of global changes in protein expression, and cell-map proteomics, the systematic study of protein-protein interactions through the isolation of protein complexes.     

Stalking metal-linked dimers

Protein dimerization is essential for cellular processes including regulation and biosignalling. While protein-protein interactions can occur through many modes, this review will focus on those interactions mediated through the binding of metal ions to the proteins. Selected techniques used to study protein-protein interactions, including size exclusion chromatography, mass spectrometry, affinity chromatography, and frontal zone chromatography, are described as applied to the characterization of the Enterococcus hirae protein CopY. CopY forms a homodimer to control the expression of proteins involved in the homeostasis of cellular copper levels. At the center of the CopY dimerization interaction lies a metal binding motif, -CxCxxxxCxC-, capable of binding Zn(II) or Cu(I). The binding of metal to this cysteine hook motif, one within each monomer, is critical to the dimerization interaction. The CopY dimer is also stabilized by hydrophobic interactions between the two monomers. The cysteine hook metal binding motif has been identified in numerous other uncharacterized proteins across the biological spectrum. The prevalence of the motif gives evidence to the biological relevance of this motif, both as a metal binding domain and as a dimerization motif.     

Quantitative model for binary measurements of protein-protein interactions.

We develop a stochastic model for quantifying the binary measurements of protein-protein interactions. A key concept in the model is the binary response function (BRF) which represents the conditional probability of successfully detecting a protein-protein interaction with a given number of the protein complexes. A popular form of the BRF is introduced and the effect of the sharpness (Hill's coefficient) of this function is studied. Our model is motivated by the recently developed yeast two-hybrid method for measuring protein-protein interaction networks. We suggest that the same phenomenological BRF can also be applied to the mass spectroscopic measurement of protein-protein interactions. Based on the model, we investigate the contributions to the network topology of protein-protein interactions from (i) the distribution of protein binary association free energy, and from (ii) the cellular protein abundance. It is concluded that the association constants among different protein pairs cannot be totally independent. It is also shown that not only the association constants but also the protein abundance could be a factor in producing the power-law degree distribution of protein-protein interaction networks.     

Two-dimensional Blue native/SDS gel electrophoresis of multi-protein complexes from whole cellular lysates: a proteomics approach

Identification and characterization of multi-protein complexes is an important step toward an integrative view of protein-protein interaction networks that determine protein function and cell behavior. The limiting factor for identifying protein complexes is the method for their separation. Blue native PAGE (BN-PAGE) permits a high-resolution separation of multi-protein complexes under native conditions. To date, BN-PAGE has only been applicable to purified material. Here, we show that dialysis permits the analysis of multi-protein complexes of whole cellular lysates by BN-PAGE. We visualized different multi-protein complexes by immunoblotting including forms of the eukaryotic proteasome. Complex dynamics after gamma interferon stimulation of cells was studied, and an antibody shift assay was used to detect protein-protein interactions in BN-PAGE. Furthermore, we identified defined protein complexes of various proteins including the tumor suppressor p53 and c-Myc. Finally, we identified multi-protein complexes via mass spectrometry, showing that the method has a wide potential for functional proteomics.