Integration of data-independent acquisition (DIA) with co-fractionation mass spectrometry (CF-MS) to enhance interactome mapping capabilities

Proteomics technologies are continually advancing, providing opportunities to develop stronger and more robust protein interaction networks (PINs). In part, this is due to the ever-growing number of high-throughput proteomics methods that are available. This review discusses how data-independent acquisition (DIA) and co-fractionation mass spectrometry (CF-MS) can be integrated to enhance interactome mapping abilities. Furthermore, integrating these two techniques can improve data quality and network generation through extended protein coverage, less missing data, and reduced noise. CF-DIA-MS shows promise in expanding our knowledge of interactomes, notably for non-model organisms (NMOs). CF-MS is a valuable technique on its own, but upon the integration of DIA, the potential to develop robust PINs increases, offering a unique approach for researchers to gain an in-depth understanding into the dynamics of numerous biological processes.     

Quantitative analysis of protein interaction network dynamics in yeast

Many cellular functions are mediated by protein–protein interaction networks, which are environment dependent. However, systematic measurement of interactions in diverse environments is required to better understand the relative importance of different mechanisms underlying network dynamics. To investigate environment‐dependent protein complex dynamics, we used a DNA‐barcode‐based multiplexed protein interaction assay in Saccharomyces cerevisiae to measure in vivo abundance of 1,379 binary protein complexes under 14 environments. Many binary complexes (55%) were environment dependent, especially those involving transmembrane transporters. We observed many concerted changes around highly connected proteins, and overall network dynamics suggested that “concerted” protein‐centered changes are prevalent. Under a diauxic shift in carbon source from glucose to ethanol, a mass‐action‐based model using relative mRNA levels explained an estimated 47% of the observed variance in binary complex abundance and predicted the direction of concerted binary complex changes with 88% accuracy. Thus, we provide a resource of yeast protein interaction measurements across diverse environments and illustrate the value of this resource in revealing mechanisms of network dynamics.     

Dynamic remodeling of Escherichia coli interactome in response to environmental perturbations

Proteins play an essential role in the vital biological processes governing cellular functions. Most proteins function as members of macromolecular machines, with the network of interacting proteins revealing the molecular mechanisms driving the formation of these complexes. Profiling the physiology-driven remodeling of these interactions within different contexts constitutes a crucial component to achieving a comprehensive systems-level understanding of interactome dynamics. Here, we apply co-fractionation mass spectrometry and computational modeling to quantify and profile the interactions of ∼2000 proteins in the bacterium Escherichia coli cultured under 10 distinct culture conditions. The resulting quantitative co-elution patterns revealed large-scale condition-dependent interaction remodeling among protein complexes involved in diverse biochemical pathways in response to the unique environmental challenges. The network-level analysis highlighted interactome-wide biophysical properties and structural patterns governing interaction remodeling. Our results provide evidence of the local and global plasticity of the E. coli interactome along with a rigorous generalizable framework to define protein interaction specificity. We provide an accompanying interactive web application to facilitate the exploration of these rewired networks.     

Interplay between binding affinity and kinetics in protein–protein interactions

To clarify the interplay between the binding affinity and kinetics of protein–protein interactions, and the possible role of intrinsically disordered proteins in such interactions, molecular simulations were carried out on 20 protein complexes. With bias potential and reweighting techniques, the free energy profiles were obtained under physiological affinities, which showed that the bound‐state valley is deep with a barrier height of 12 ? 33 RT. From the dependence of the affinity on interface interactions, the entropic contribution to the binding affinity is approximated to be proportional to the interface area. The extracted dissociation rates based on the Arrhenius law correlate reasonably well with the experimental values (Pearson correlation coefficient R = 0.79). For each protein complex, a linear free energy relationship between binding affinity and the dissociation rate was confirmed, but the distribution of the slopes for intrinsically disordered proteins showed no essential difference with that observed for ordered proteins. A comparison with protein folding was also performed. Proteins 2016; 84:920–933. © 2016 Wiley Periodicals, Inc.     

A common neighbor based technique to detect protein complexes in PPI networks

Detection of protein complexes by analyzing and understanding PPI networks is an important task and critical to all aspects of cell biology. We present a technique called PROtein COmplex DEtection based on common neighborhood (PROCODE) that considers the inherent organization of protein complexes as well as the regions with heavy interactions in PPI networks to detect protein complexes. Initially, the core of the protein complexes is detected based on the neighborhood of PPI network. Then a merging strategy based on density is used to attach proteins and protein complexes to the core-protein complexes to form biologically meaningful structures. The predicted protein complexes of PROCODE was evaluated and analyzed using four PPI network datasets out of which three were from budding yeast and one from human. Our proposed technique is compared with some of the existing techniques using standard benchmark complexes and PROCODE was found to match very well with actual protein complexes in the benchmark data. The detected complexes were at par with existing biological evidence and knowledge.     

溶酶体相关细胞器生物发生复合体相互作用组构成胞内体运输网络

随着高等生物中十几个新的参与囊泡运输的 Hermansky-Pudlak 综合征(HPS)蛋白质的发现, 认为可能存在一类新的囊泡运输通路。该通路主要由新近鉴定的 3 个被称为溶酶体相关细胞器生物发生复合体(BLOC)所组成, 被分别命名为BLOC-1、BLOC-2 和 BLOC-3。越来越多的证据表明这些复合体与以前认识较清楚的 AP3 和 HOPS 复合体共同在胞内体运输中起重要作用。这些复合体之间的相互作用构成了以胞内体和细胞骨架为连接纽带的参与蛋白质运输的复杂网络。该网络中的每个节点的相互作用可区分为复合体内和复合体外相互作用两大类。复合体之间的联系可以是来自不同复合体亚基间的直接相互作用, 也可以通过耦联的节点联结不同的复合体。解析这一复杂网络有助于进一步了解参与蛋白质和膜运输这一动态而精细网络的结构与功能。一旦该网络结构得到破坏, 则可能导致如 HPS 这类囊泡运输或细胞器发生障碍性疾病。     

Genes2Networks: connecting lists of gene symbols using mammalian protein interactions databases

Background  

In recent years, mammalian protein-protein interaction network databases have been developed. The interactions in these databases are either extracted manually from low-throughput experimental biomedical research literature, extracted automatically from literature using techniques such as natural language processing (NLP), generated experimentally using high-throughput methods such as yeast-2-hybrid screens, or interactions are predicted using an assortment of computational approaches. Genes or proteins identified as significantly changing in proteomic experiments, or identified as susceptibility disease genes in genomic studies, can be placed in the context of protein interaction networks in order to assign these genes and proteins to pathways and protein complexes.     

Nanoscale protein domain motion and long-range allostery in signaling proteins—a view from neutron spin echo spectroscopy

Many cellular proteins are multi-domain proteins. Coupled domain–domain interactions in these multidomain proteins are important for the allosteric relay of signals in the cellular signaling networks. We have initiated the application of neutron spin echo spectroscopy to the study of nanoscale protein domain motions on submicrosecond time scales and on nanometer length scale. Our NSE experiments reveal the activation of protein domain motions over a long distance of over more than 100 Å in a multidomain scaffolding protein NHERF1 upon binding to another protein, Ezrin. Such activation of nanoscale protein domain motions is correlated with the allosteric assembly of multi-protein complexes by NHERF1 and Ezrin. Here, we summarize the theoretical framework that we have developed, which uses simple concepts from nonequilibrium statistical mechanics to interpret the NSE data, and employs a mobility tensor to describe nanoscale protein domain motion. Extracting nanoscale protein domain motion from the NSE does not require elaborate molecular dynamics simulations, nor complex fits to rotational motion, nor elastic network models. The approach is thus more robust than multiparameter techniques that require untestable assumptions. We also demonstrate that an experimental scheme of selective deuteration of a protein subunit in a complex can highlight and amplify specific domain dynamics from the abundant global translational and rotational motions in a protein. We expect NSE to provide a unique tool to determine nanoscale protein dynamics for the understanding of protein functions, such as how signals are propagated in a protein over a long distance to a distal domain.     

New approach to evaluating the effects of a drug on protein complexes with quantitative proteomics,using the SILAC method and bioinformatic approach

ABSTRACT

Protein–protein interactions (PPIs) lead the formation of protein complexes that perform biochemical reactions that maintain the living state of the living cell. Although therapeutic drugs should influence the formation of protein complexes in addition to PPI network, the methodology analyzing such influences remain to be developed. Here, we demonstrate that a new approach combining HPLC (high performance liquid chromatography) for separating protein complexes, and the SILAC (stable isotope labeling using amino acids in cell culture) method for relative protein quantification, enable us to identify the protein complexes influenced by a drug. We applied this approach to the analysis of thalidomide action on HepG2 cells, assessed the identified proteins by clustering data analyses, and assigned 135 novel protein complexes affected by the drug. We propose that this approach is applicable to elucidating the mechanisms of actions of other therapeutic drugs on the PPI network, and the formation of protein complexes.     

Discovering implicit protein–protein interactions in the <Emphasis Type="Italic">Cell Cycle</Emphasis> using bioinformatics approaches

The cell division control protein (Cdc2) kinase is a catalytic subunit of a protein kinase complex, called the M phase promoting factor, which induces entry into mitosis and is universal among eukaryotes. This protein is believed to play a major role in cell division and control. The lives of biological cells are controlled by proteins interacting in metabolic and signaling pathways, in complexes that replicate genes and regulate gene activity, and in the assembly of the cytoskeletal infrastructure. Our knowledge of protein–protein (P–P) interactions has been accumulated from biochemical and genetic experiments, including the widely used yeast two-hybrid test. In this paper we examine if P–P interactions in regenerating tissues and cells of the anuran Xenopus laevis can be discovered from biomedical literature using computational and literature mining techniques. Using literature mining techniques, we have identified a set of implicitly interacting proteins in regenerating tissues and cells of Xenopus laevis that may interact with Cdc2 to control cell division. Genome sequence based bioinformatics tools were then applied to validate a set of proteins that appear to interact with the Cdc2 protein. Pathway analysis of these proteins suggests that Myc proteins function as the regulator of M phase initiation by controlling expression of the Akt1 molecule that ultimately inhibits the Cdc2-cyclin B complex in cells. P–P interactions that are implicitly appearing in literature can be effectively discovered using literature mining techniques. By applying evolutionary principles on the P–P interacting pairs, it is possible to quantitatively analyze the significance of the associations with biological relevance. The developed BioMap system allows discovering implicit P–P interactions from large quantity of biomedical literature data. The unique similarities and differences observed within the interacting proteins can lead to the development of the new hypotheses that can be used to design further laboratory experiments.     

Protein–protein interaction network analysis of insecticide resistance molecular mechanism in Drosophila melanogaster

The problem of resistance has not been solved fundamentally at present, because the development speed of new insecticides can not keep pace with the development speed of resistance, and the lack of understanding of molecular mechanism of resistance. Here we collected seed genes and their interacting proteins involved in insecticide resistance molecular mechanism in Drosophila melanogaster by literature mining and the String database. We identified a total of 528 proteins and 13514 protein–protein interactions. The protein interaction network was constructed by String and Pajek, and we analyzed the topological properties, such as degree centrality and eigenvector centrality. Proteasome complexes and drug metabolism—cytochrome P450 were an enrichment by Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. This is the first time to explore the insecticide resistance molecular mechanism of D. melanogaster by the methods and tools of network biology, it can provide the bioinformatic foundation for further understanding the mechanisms of insecticide resistance.     

Structure-based binding between protein farnesyl transferase and PRL-PTP of malaria parasite: an interaction study of prenylation process in Plasmodium

Protein prenylation is a post-translational modification critical for many cellular processes such as DNA replication, signaling, and trafficking. It is mediated by protein farnesyltransferase by recognizing ‘CAAX’ motif on protein substrate. Plasmodium falciparum also possesses many such proteins with ‘CAAX’ motif, involved in various pathways of the parasite. The interaction studies of PfPFT with its substrate were carried out using synthetic peptides but not with full protein. Therefore, in this study, we have modeled both PfPFT and its substrate protein tyrosine phosphatase (PfPRL-PTP) followed by interaction studies using protein–protein docking and molecular dynamics simulation. Our findings provided a clear picture of interactions at atomic level between prenyltransferase and its protein substrate. We are assured that this piece of information can be extended to many other proteins of parasite containing ‘CAAX’ motif and that it may also lead to the development of anti-malarials based on the inhibition of prenylation-dependent pathways of parasite..     

In vivo assay for protein-protein interactions using Drosophila chromosomes

The ability of a chimeric HP1-Polycomb (Pc) protein to bind both to heterochromatin and to euchromatic sites of Pc protein binding was exploited to detect stable protein-protein interactions in vivo. Previously, we showed that endogenous Pc protein was recruited to ectopic heterochromatic binding sites by the chimeric protein. Here, we examine the association of other Pc group (Pc-G) proteins. We show that Posterior sex combs (Psc) protein also is recruited to heterochromatin by the chimeric protein, demonstrating that Psc protein participates in direct protein-protein interaction with Pc protein or Pc-associated protein. In flies carrying temperature-sensitive alleles of Enhancer of zeste[E(z)] the general decondensation of polytene chromosomes that occurs at the restrictive temperature is associated with loss of binding of endogenous Pc and chimeric HP1-Polycomb protein to euchromatin, but binding of HP1 and chimeric HP1-Polycomb protein to the heterochromatin is maintained. The E(z) mutation also results in the loss of chimera-dependent binding to heterochromatin by endogenous Pc and Psc proteins at the restrictive temperature, suggesting that interaction of these proteins is mediated by E(z) protein. A myc-tagged full-length Suppressor 2 of zeste [Su(z)2] protein interacts poorly or not at all with ectopic Pc-G complexes, but a truncated Su(z)2 protein is strongly recruited to all sites of chimeric protein binding. Trithorax protein is not recruited to the heterochromatin by the chimeric HP1-Polycomb protein, suggesting either that this protein does not interact directly with Pc-G complexes or that such interactions are regulated. Ectopic binding of chimeric chromosomal proteins provides a useful tool for distinguishing specific protein-protein interactions from specific protein-DNA interactions important for complex assembly in vivo.     

Analytical Guidelines for co-fractionation Mass Spectrometry Obtained through Global Profiling of Gold Standard Saccharomyces cerevisiae Protein Complexes

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Highlights

• •Guidelines for studying protein complexes via co-fractionation mass spectrometry.
• •A novel procedure for profiling gold standard protein complexes in CF-MS data.
• •Recommendations for efficient CF-MS fractionation collection.
• •Scoring metric recommendations for precise and sensitive CF-MS data analysis.

     

Distribution, lateral mobility and function of membrane proteins incorporated into giant unilamellar vesicles

GUVs have been widely used for studies on lipid mobility, membrane dynamics and lipid domain (raft) formation, using single molecule techniques like fluorescence correlation spectroscopy. Reports on membrane protein dynamics in these types of model membranes are by far less advanced due to the difficulty of incorporating proteins into GUVs in a functional state. We have used sucrose to prevent four distinct membrane protein(s) (complexes) from inactivating during the dehydration step of the GUV-formation process. The amount of sucrose was optimized such that the proteins retained 100% biological activity, and many proteo-GUVs were obtained. Although GUVs could be formed by hydration of lipid mixtures composed of neutral and anionic lipids, an alternate current electric field was required for GUV formation from neutral lipids. Distribution, lateral mobility, and function of an ATP-binding cassette transport system, an ion-linked transporter, and a mechanosensitive channel in GUVs were determined by confocal imaging, fluorescence correlation spectroscopy, patch-clamp measurements, and biochemical techniques. In addition, we show that sucrose slows down the lateral mobility of fluorescent lipid analogs, possibly due to hydrogen-bonding with the lipid headgroups, leading to larger complexes with reduced mobility.     

The mitochondrial complexome of Arabidopsis thaliana

Mitochondria are central to cellular metabolism and energy conversion. In plants they also enable photosynthesis through additional components and functional flexibility. A majority of those processes relies on the assembly of individual proteins to larger protein complexes, some of which operate as large molecular machines. There has been a strong interest in the makeup and function of mitochondrial protein complexes and protein–protein interactions in plants, but the experimental approaches used typically suffer from selectivity or bias. Here, we present a complexome profiling analysis for leaf mitochondria of the model plant Arabidopsis thaliana for the systematic characterization of protein assemblies. Purified organelle extracts were separated by 1D Blue native (BN) PAGE, a resulting gel lane was dissected into 70 slices (complexome fractions) and proteins in each slice were identified by label free quantitative shot‐gun proteomics. Overall, 1359 unique proteins were identified, which were, on average, present in 17 complexome fractions each. Quantitative profiles of proteins along the BN gel lane were aligned by similarity, allowing us to visualize protein assemblies. The data allow re‐annotating the subunit compositions of OXPHOS complexes, identifying assembly intermediates of OXPHOS complexes and assemblies of alternative respiratory oxidoreductases. Several protein complexes were discovered that have not yet been reported in plants, such as a 530 kDa Tat complex, 460 and 1000 kDa SAM complexes, a calcium ion uniporter complex (150 kDa) and several PPR protein complexes. We have set up a tailored online resource ( https://complexomemap.de/at_mito_leaves ) to deposit the data and to allow straightforward access and custom data analyses.     

Proximity probing assays for simultaneous visualization of protein complexes in situ

Evaluation of: Leuchowius KJ, Clausson CM, Grannas K et al. Parallel visualization of multiple protein complexes in individual cells in tumor tissue. Mol. Cell Proteomics doi:10.1074/mcp.O112.023374 (2013) (Epub ahead of print).

Techniques for in situ detection and quantification of proteins in fixed tissue remain an important element of both basic biological analyses and clinical biomarker research. The practical importance of such techniques can be exemplified by the everyday clinical use of immunohistochemical detection of the estrogen receptor and HER2 in tissues from breast cancer patients. Several techniques are currently available for detection of single proteins and post-translational modifications, but very few are suitable for detection of protein complexes. Methods that enable simultaneous detection and quantification of protein complexes provide novel possibilities for understanding the biological role(s) of protein complexes and may open new opportunities to improve clinical biomarker research. Leuchowius et al. describe an improved proximity ligation assay for in situ detection of protein complexes, which is able to detect and quantify several protein complexes simultaneously in the same tissue specimen.