Protein-Protein Interactions: Methods and Protocols (Methods in Molecular Biology, 2690)

Description

This detailed volume provides a comprehensive collection of classic and cutting-edge methods and techniques in mapping protein-protein interactions. The chapters include a variety of in vitro and in vivo experimental methods covering cell biology, biochemistry, and biophysics. In addition, the book also explores in silico methods including sequence-, structure-, and phylogenetic profile-based approaches as well as gene expression and machine learning methods. Written for the highly successful Methods in Molecular Biology series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step and readily reproducible laboratory protocols, as well as tips on troubleshooting and avoiding known pitfalls.  Authoritative and practical, Protein-Protein Interactions: Methods and Protocols serves as an ideal guide for researchers working in protein science and beyond. Preface Contents Contributors Chapter 1: Yeast Two-Hybrid Technique to Identify Protein-Protein Interactions 1 Introduction 2 Materials 2.1 Yeast Two-Hybrid Library Construction 2.2 Constructs for Pairwise Y2H 2.3 Yeast Transformation 2.4 Y2H Library Screening 3 Methods 3.1 Yeast Two-Hybrid Library Construction 3.2 Yeast Transformation 3.3 Testing Autoactivation 3.4 Yeast Two-Hybrid Library Screening 3.5 Pairwise Y2H Screening 4 Notes References Chapter 2: Cytotrap: An Innovative Approach for Protein-Protein Interaction Studies for Cytoplasmic Proteins 1 Introduction 2 Materials 2.1 Reagents and Supply Items 2.2 Specialist Equipment 2.3 The Composition of All Buffers, Media, and Solutions 3 Methods 3.1 Processing the Yeast Strain and Glycerol Stock Preparation Process (Fig. 2) 3.2 Construction of the Bait-hSos Protein Fusion and Target Plasmid 3.3 Competent Cell Preparation for cdc25H Yeast Strain 3.4 Transformation of the Plasmids into Yeast and Identification of the Interaction System 3.5 Confirmation of the Interaction Between Bait Protein and Target Protein Using a Co-transformation Approach 3.6 Confirmation of the Interaction Between Bait Protein and Target Protein Using Yeast Mating System 4 Notes References Chapter 3: Analyzing Protein-Protein Interactions Using the Split-Ubiquitin System 1 Introduction 1.1 Yeast mbSUS Reporter System 1.2 mbSUS Assays for Analysis of Tripartite Interactions 2 Materials 2.1 Yeast SUS Assays Chemicals List 2.2 Antibodies 2.3 mbSUS Yeast Strains 2.4 The Reagents and Media for Yeast Growth and Transformation. 2.5 Yeast Selection Media for mbSUS, GPS, and Tri-SUS Systems 2.6 The Amino Acid and Nucleobase Stock Solutions for mbSUS, GPS, and Tri-SUS Systems (see Note 3) 3 Methods 3.1 Protocol for Yeast Transformation and Selection for mbSUS, GPS, and Tri-SUS Assays (See Note 4) 3.2 The Steps for the Protocol for Yeast Growth Assay (See Note 4 and Fig. 3) 4 Notes References Chapter 4: Detection of Protein-Protein Interactions Utilizing the Split-Ubiquitin Membrane-Based Yeast Two-Hybrid System 1 Introduction 2 Materials 3 Methods 3.1 Generation of NMY51 Yeast Strain Expressing the Functional Bait Protein 3.1.1 Transformation of the Bait Construct into NMY51 3.1.2 Bait Protein Expression Check by Western Blot 3.1.3 Bait Functional Assay 3.2 Standardization of Screening Stringency Using a Pilot Transformation 3.3 Library Screening and Confirmation of Interaction 3.3.1 Library Transformation 3.3.2 Recovery of Prey Plasmid 3.3.3 Bait-Dependency Test 3.3.4 Qualitative ß-Galactosidase Activity (Colony-Lift Filter Assay) 4 Notes References Chapter 5: Preparation and Utilization of a Versatile GFP-Protein Trap-Like System for Protein Complex Immunoprecipitation in ... 1 Introduction 2 Materials 2.1 Plant Material and Growth Conditions 2.2 Agrobacterium culture 2.3 Agrobacterium Infiltration Buffer 2.4 Protein Extraction Buffer 2.5 Lysate Dilution Buffer 2.6 Antibody 2.7 Antibody Dilution Buffer 2.8 Magnetic Beads 2.9 Bead Washing Buffer 2.10 Elution and Sample Loading Buffer 3 Methods 3.1 Agrobacterium Co-infiltration 3.2 Sample Preparation 3.3 Magnetic Bead Preparation 3.4 Antibody Dilution and Binding 3.5 Sample Loading and Protein Complex Immunoprecipitation 3.6 Bead Washing 4 Notes References Chapter 6: Tandem Affinity Purification (TAP) of Interacting Prey Proteins with FLAG- and HA-Tagged Bait Proteins 1 Introduction 2 Materials 2.1 Cell Transfection and Harvest 2.2 Cell Lysis 2.3 Affinity Purification 2.3.1 FLAG IP 2.3.2 HA IP 2.4 SDS-PAGE 3 Methods 3.1 Cell Transfection and Harvest 3.2 Cell Lysis 3.3 Affinity Purification 3.3.1 Column Packing in FLAG IP 3.3.2 Sample Loading in FLAG IP 3.3.3 Washing in FLAG IP 3.3.4 Elution in FLAG IP 3.3.5 HA IP 3.4 SDS-PAGE 4 Notes References Chapter 7: Affinity Purification-Mass Spectroscopy (AP-MS) and Co-Immunoprecipitation (Co-IP) Technique to Study Protein-Prote... 1 Introduction 2 Materials 2.1 AP-MS 2.2 Co-IP 3 Methods 3.1 AP-MS 3.2 Co-IP 4 Notes References Chapter 8: Co-immunoprecipitation-Based Identification of Effector-Host Protein Interactions from Pathogen-Infected Plant Tiss... 1 Introduction 2 Materials 3 Methods 3.1 Experimental Design 3.1.1 Selection of Promoter of Interest for the Co-IP 3.1.2 Selection of Epitope Tag for the Co-IP 3.1.3 Negative Control 3.2 Preparation of DNA for Protoplast Transformations 3.3 Preparation of Ustilago maydis FB1 and FB2 Competent Cells and Their Transformation with Plasmid DNA 3.4 Growing Maize Seedlings for Infection 3.5 Infection of Maize Seedlings with FB1 and FB2 Strains 3.6 Co-immunoprecipitation of an Effector-Target Protein Complex from Infected Maize 3.6.1 Immunoprecipitation of Protein Complexes Using Agarose Beads 3.6.2 Immunoprecipitation of Protein Complexes Using Magnetic Beads 4 Notes References Chapter 9: Co-immunoprecipitation for Assessing Protein-Protein Interactions in Agrobacterium-Mediated Transient Expression Sy... 1 Introduction 2 Materials 2.1 Plant Material and Agrobacterium tumefaciens 2.2 Equipment 2.3 Reagents 3 Methods 3.1 Transient Expression in Nicotiana benthamiana 3.2 Preparing Protein Extract and Co-IP 3.3 Immunoblotting Analysis 4 Notes References Chapter 10: Detection of Protein-Protein Interactions Using Glutathione-S-Transferase (GST) Pull-Down Assay Technique 1 Introduction 2 Materials 2.1 Protein Expression and Purification 2.2 In Vitro GST Pull-Down 2.3 Detection of Interacting Proteins 3 Methods 3.1 Protein Expression and Purification 3.2 In Vitro GST Pull-Down 3.3 Detection of Interacting Proteins 4 Notes References Chapter 11: Bimolecular Fluorescence Complementation (BiFC) Assay to Visualize Protein-Protein Interactions in Living Cells 1 Introduction 2 Materials 3 Methods 4 Notes References Chapter 12: Detecting Protein-Protein Interactions Using Bimolecular Fluorescence Complementation (BiFC) and Luciferase Comple... 1 Introduction 2 Materials 2.1 Common Materials for BiFC and LCA 2.2 Materials Specific to BiFC 2.3 Materials Specific to LCA 3 Methods 3.1 Cloning 3.2 Infiltration of Nicotiana benthamiana Leaves 3.3 Imaging BiFC Samples 3.4 Detection of LCA 3.4.1 Visualization of LCA in Leaf 3.4.2 Quantification of Protein Interaction Through Measuring Luciferase Activity 3.4.3 Data Analysis 4 Notes References Chapter 13: Forster Resonance Energy Transfer (FRET) to Visualize Protein-Protein Interactions in the Plant Cell 1 Introduction 2 Materials 3 Methods 4 Notes References Chapter 14: Dynamic Proximity Tagging in Living Plant Cells with Pupylation-Based Interaction Tagging 1 Introduction 2 Materials 2.1 Plant Material and Growth Conditions 2.2 Construction of the Plant Expression Plasmids 2.3 Protoplast Isolation and Transfection 2.4 Immunoprecipitation 2.5 Immunoblotting or Mass Spectrometry Analysis 3 Methods 3.1 Protoplast Isolation 3.2 Protoplast Transfection 3.3 Immunoprecipitation 3.4 Immunoblotting Analysis 3.5 Prepare Samples for Mass Spectrometry Analysis 4 Notes References Chapter 15: Characterization of Small Molecule-Protein Interactions Using SPR Method 1 Introduction 2 Materials 3 Methods 3.1 Preparation for Immobilization (See Note 6) 3.2 Immobilization of Ligand Using Amine Coupling 3.3 Kinetics/Affinity Measurements (See Note 16) 3.4 Kinetics/Affinity Data Analysis 4 Notes References Chapter 16: High-Throughput Protein-Protein Interactions Screening Using Pool-Based Liquid Yeast Two-Hybrid Pipeline 1 Introduction 2 Materials 2.1 Reagents 2.2 Yeast Strains, Plasmids, and Primers 2.3 Media and Solutions 2.4 Disposables 2.5 Equipment 3 Methods 3.1 Generation of Expression Plasmids by Recombinational Cloning 3.2 Bacterial Transformation 3.3 Bacterial Culture PCR 3.4 Yeast Transformation 3.5 Yeast Cell Lysis 3.6 Yeast Lysate PCR 3.7 Identification of Autoactivating DB-X Hybrid Proteins 3.8 Construction of DB Baits Pool 3.9 AD-Collection Culture 3.10 Mating 3.11 Screening 3.12 AD-Interacting Proteins Rearray and Archival Stock 3.13 Deconvolution and Targeted Matricial Assay Cultures 3.14 Mating 3.15 Screening 3.16 Diploids Rearray and Interacting Proteins Identification 4 Notes References Chapter 17: Dynamic Enrichment for Evaluation of Protein Networks (DEEPN): A High Throughput Yeast Two-Hybrid (Y2H) Protocol t... 1 Introduction 2 Materials 2.1 Y2H Library Construction 2.2 Antibodies 2.3 Special Equipment 2.4 Composition of All Buffers, Media, and Solutions 2.4.1 Synthetic Defined (SD) Media for Yeast 2.4.2 Yeast-Rich Media (YPD) 2.4.3 Buffered Yeast-Rich Media (bYPDA) 2.4.4 TWIRL Sample Buffer 3 Methods 3.1 Development of Plasmids Containing Gal4-DNA-Binding Domain 3.2 Expression of Fusion Proteins Containing Gal4 DNA-Binding Domain 3.3 Self-Activation Test 3.3.1 Test Selection Conditions for Yeast Two-Hybrid (Y2H) Interaction 3.3.2 Mating and Selection 3.4 Preparation of DEEPN Samples for Illumina Sequencing 3.4.1 DNA Extraction/Purification 3.4.2 PCR Purification of cDNA Fragments 3.4.3 Illumina Sequencing 3.5 DEEPN 4 Notes References Chapter 18: An Interactome Assay for Detecting Interactions between Extracellular Domains of Receptor Kinases 1 Introduction 2 Materials 2.1 Secreted Expression of Extracellular Domains 2.2 Cell Surface Interaction Assay 3 Methods 3.1 Secreted Expression of Extracellular Domains 3.2 Cell Surface Interaction Assay 4 Notes References Chapter 19: Next-Generation Yeast Two-Hybrid Screening to Discover Protein-Protein Interactions 1 Introduction 1.1 Considerations for the cDNA Library 1.2 Choose Your Y2H System 1.3 Watch for Autoactivators 1.4 Protocol Overview 2 Materials 2.1 Library-Scale Mating of Bait Strain with Prey Library 2.2 Enrich Diploids in Liquid Culture (SC-LW1) 2.3 Sub-Culture Yeast Populations in Diploid (SC-LW2) and Interactor Media (SC-LWH1) 2.4 Harvest SC-LW2 Cell Population 2.5 Monitor SC-LWH1 Cultures and Sub-Culture in SC-LWH2 Media 2.6 Monitor SC-LWH2 Cultures and Harvest Cells 2.7 Plasmid Isolation from Yeast 2.8 PCR Amplification of Prey Sequences 3 Methods 3.1 Library-Scale Mating of Bait Strain with Prey Library 3.2 Enrich Diploids in SC-LW1 Media 3.3 Sub-Culture into Diploid (SC-LW2) and Interactor Media (SC-LWH1) 3.4 Monitor SC-LW2 Cultures and Harvest Cells Once OD600 > 2.5 3.5 Monitor SC-LWH1 Cultures and Subculture Cells into SC-LWH2 Media Once OD600 > 2.5 3.6 Monitor SC-LWH2 Cultures and Harvest Cells Once OD600 > 2.5 3.7 Yeast Plasmid Isolation 3.8 PCR Amplification of Prey Sequences 4 Notes References Chapter 20: Bioinformatic Analysis of Yeast Two-Hybrid Next-Generation Interaction Screen Data 1 Introduction 2 Materials 2.1 Computational Hardware 2.2 Computational Software 3 Methods 3.1 Design the Y2H-NGIS Experiment 3.1.1 Identify Available Resources for Your Experiment 3.1.2 Select Number of Baits and Preys 3.1.3 Select Number of Replicates, Sequencing Depth, and Controls 3.2 Generate the Count Data Using the NGPINT V2 Pipeline 3.2.1 Prepare Inputs to the NGPINT V2 Pipeline 3.2.2 Populate the Configuration File 3.2.3 Select Primer Sequences for Fusion Read Identification 3.2.4 Run NGPINT V2 and Identify the Outputs 3.3 Rank Interactions with the Y2H-SCORES Software 3.3.1 File Structure 3.3.2 Software Testing 3.3.3 Count Datasets 3.3.4 Normalization 3.3.5 Select Parameters and Run Y2H-SCORES 3.3.6 Identify the Y2H-SCORES Outputs 4 Notes References Chapter 21: Discovering Protein-Protein Interactions using Co-Fractionation-Mass Spectrometry with Label-Free Quantitation 1 Introduction 2 Materials 2.1 Cell/Tissue Lysis 2.2 Protein Complex Isolation by SEC 2.3 Sample Preparation for Mass Spectrometry Analysis 2.4 LC-MS/MS 2.5 Protein Identification and Quantitation 2.6 Protein-Protein Interaction Prediction 3 Methods 3.1 Tissue/Cell Lysis and Protein Complex Enrichment 3.2 Protein Complex Isolation by SEC 3.3 Preparation of Fractionated Protein Samples for MS Analysis 3.4 Protein Identification and Quantitation 3.5 Protein-Protein Interaction Prediction with PrInCE 4 Notes References Chapter 22: Protein-Protein Interaction Network Mapping by Affinity Purification Cross-Linking Mass Spectrometry (AP-XL-MS) ba... 1 Introduction 2 Materials 2.1 Cell Culture 2.2 Cell Treatments 2.3 Affinity Purification 2.4 SDS Polyacrylamide Gel 2.5 Digestion 2.6 Sample Analysis (Nano ESI-LC-MS/MS) 2.7 Dual Cleavable Cross-Linking Technology (DUCCT) and Other Cross-Linkers 2.8 Data Analysis 3 Methods 3.1 Cell Culture 3.1.1 HA-Tagged HEK293 Cell Culture 3.2 Cell Treatments 3.2.1 Simvastatin Treatment of HA-Tagged TLR2-MD2-CD14 Cells 3.2.2 LPS-Biotin Treatment of HA-Tagged TLR2-MD2-CD14 Cells 3.2.3 Cross-Linker 3.3 Cell Lysis 3.4 Affinity Purification 3.4.1 Biotin Avidin Affinity Purification 3.4.2 Fusion-Tag Purification 3.5 Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) 3.6 Digestion 3.6.1 In-Solution Digestion 3.6.2 In-Gel Digestion 3.7 Sample Analysis (Nano ESI-LC-MS/MS) 3.8 Data Analysis 4 Notes References Chapter 23: Protein Interaction Screen on a Peptide Matrix (PrISMa) 1 Introduction 2 Materials 2.1 Peptide Array Setup 2.2 Pull-down Assay and Digestion 2.3 Liquid Chromatography Coupled Mass Spectrometry (LC-MS) 2.4 Equipment Setup 3 Methods 3.1 Parallel Peptide Pull-Down Assay and Preparation of the Spots for Mass Spectrometric Analysis (See Note 2) 3.2 Digestion of the Bound Material (See Note 4) 3.3 LC-MS (See Note 8) 3.4 Data Analysis 4 Notes References Chapter 24: Analyzing Protein Interactions by MAC-Tag Approaches 1 Introduction 2 Materials 2.1 Cloning 2.2 Generation of Stable Cell Lines 2.3 Affinity Purification 2.4 Proximity Labeling 2.5 Sample Preparation for Mass Spectrometry 3 Methods 3.1 Generation of MAC/MAC2/MAC3-Tagged Gene of Interest Using Gateway Cloning 3.2 Generation of Stable Cell Lines Expressing the MAC/MAC2/MAC3-Tagged POI 3.3 Generation of Cell Pellets for Protein Purification 3.3.1 For Affinity Purification 3.3.2 For Proximity Labeling 3.3.3 For Proximity Labeling with Activation of Kinases Using Pervanadate 3.4 Protein Purification 3.4.1 For Affinity Purification 3.4.2 For Proximity Labeling 3.5 Sample Preparation for Mass Spectrometry 3.6 Mass Spectrometry and Data Analysis 4 Notes References Chapter 25: Identification and Quantification of Affinity-Purified Proteins with MaxQuant, Followed by the Discrimination of N... 1 Introduction 2 Materials 2.1 Trypsin Digestion 2.2 Sep-Pak Cleanup 2.3 UPLC-MS/MS 2.4 MaxQuant 2.5 CRAPome 3 Methods 3.1 Recovery of Immunoprecipitated Proteins and Trypsin Digestion 3.2 Sep-Pak Cleanup 3.3 UPLC-MS/MS 3.4 Computational Analysis of LC-MS/MS Data with MaxQuant 3.5 Post-Acquisition Data Analysis with CRAPome 3.5.1 CRAPome 2.0 The CRAPome Analysis Pipeline 4 Notes References Chapter 26: Cataloguing Protein Complexes In Planta Using TurboID-Catalyzed Proximity Labeling 1 Introduction 2 Materials 2.1 Bacterial Strains 2.2 Vectors 2.3 General Materials and Equipment 2.4 Hairy Root Transformation 2.4.1 Reagents 2.4.2 Materials and Equipment 2.5 Induction of Bait-TurboID Expression, Protein Extraction, and Expression Analysis Using Immunoblotting 2.5.1 Reagents 2.5.2 Materials and Equipment 2.6 Sample Preparation for Mass Spectrometry 2.6.1 Reagents, Solutions, and Buffers 2.6.2 Materials and Equipment 2.7 LC-MS/MS 2.8 Data Analysis 3 Methods 3.1 Vector Construction for Estradiol-Inducible Bait-TurboID Expression in Rhizogenic Agrobacterium-Transformed Hairy Roots 3.2 Hairy Root Transformation, Selection, and Cultivation of Transformed Hairy Roots 3.2.1 Seed Sterilization and Germination (7-10 Days) 3.2.2 Growing A. rhizogenes Strain ATCC15834 (Day -1) 3.2.3 Hairy Root Transformation (Day 1) 3.2.4 Selection and Root Induction (Day 5) 3.2.5 Subculturing of Hairy Roots (Week 4-8) 3.3 Immunoblot Analysis of Bait-TurboID Expression in Transformed Hairy Roots 3.4 Sample Preparation for MS 3.4.1 Protein Extraction and Streptavidin Beads Enrichment (Day 1) 3.4.2 Trypsin Digestion (Day 2) 3.4.3 Peptide Isolation (Day 3) 3.4.4 Peptide Purification (Day 4) 3.4.5 Quality Assessment of TurboID MS Sample Preparation by Immunoblot Analysis 3.5 LC-MS/MS Analysis 3.6 MS-Data Analysis 4 Notes References Chapter 27: A Data-Driven Signaling Network Inference Approach for Phosphoproteomics 1 Introduction 2 Materials 3 Methods 3.1 R Package for Phosphoproteomic Analysis, Visualization, and Signaling Network Inference 3.2 Detailed Source Code for Phosphoproteomic Analysis and Signaling Network Inference 3.2.1 Analyzing Label-Free Phosphoproteomics from MaxQuant Searches Box 1 Loading and filtering label-free phosphoproteomics data from MaxQuant searches Box 2 Subset creation 3.2.2 Normalization and Statistical Analysis Box 3 Normalizing the data using variance stabilization normalization (vsn) Box 4 Performing statistical analysis by fitting a linear mixed model 3.2.3 Identify Regulator-Target Pairs for Network Inference Box 5 Identifying kinases/phosphatases within the data Box 6 Scaling and discretizing the phosphorylation intensities Box 7 Identifying all possible regulator-target pairs 3.2.4 Network Inference Through Dynamic Bayesian Scoring Box 8 Computing the regulator-target interaction probabilities with the BDeu score Box 9 Identifying the sign of each inferred interaction (phosphorylation or dephosphorylation) 4 Notes References Chapter 28: Pairwise and Multi-chain Protein Docking Enhanced Using LZerD Web Server 1 Introduction 1.1 AttentiveDist Protein Structure Prediction Algorithm 1.2 The LZerD Pairwise Docking Algorithm 1.3 Multi-LZerD Multiple Chain Docking Algorithm 2 Materials 2.1 Computational Requirements 3 Method 3.1 Creating an Account in the LZerD Web Server 3.2 Using AttentiveDist 3.3 Using LZerD for Pairwise Docking 3.4 Constraints to Restrict the Docking 3.5 Homodimeric Docking 3.6 Using Multi-LZerD for Multi-chain Docking 3.7 Output of Docking 4 Notes References Chapter 29: Predicting Protein Interaction Sites Using PITHIA 1 Introduction 2 Materials 2.1 Source Code 2.2 Installation 2.3 Computing Environment 2.4 Input Formatting 2.5 Feature Extraction 3 Methods 3.1 MSA Size 3.2 Padding 3.3 Implementation Limitations 3.4 Architecture 3.5 Availability 4 Notes References Chapter 30: Using PlaPPISite to Predict and Analyze Plant Protein-Protein Interaction Sites 1 Introduction 2 Materials 2.1 The Environment of Database and System Requirements 2.2 The Input Data 2.3 The Datasets Deposited in PlaPPISite 3 Methods 3.1 Searching for a Single Protein 3.2 Search for an Interaction Pair 3.3 Prediction of Complex Structure 4 Notes References Chapter 31: Machine Learning Methods for Virus-Host Protein-Protein Interaction Prediction 1 Introduction 2 Materials 2.1 Computing Environment 2.2 Operating Systems and Its Specifications 2.3 Scripting Language/Software 2.4 Packages/Tools 3 Methods 3.1 Set Up the Environment 3.2 Compile the Dataset 3.3 Construct the Feature Vector 3.4 Apply Ensemble Techniques and Generate the Model 3.5 Evaluate the Performance 4 Notes References Chapter 32: Protein-Protein Interaction Network Exploration Using Cytoscape 1 Introduction 2 Materials 2.1 Fetching the AI-1MAIN Dataset from Plant Interactome Database 2.2 System Requirements 3 Methods 3.1 Data Acquisition 3.2 Set Up Cytoscape: Download and Installation 3.3 Import Network Data in Cytoscape 3.4 Network Customization: Style and Layouts 3.5 Network Clustering 3.6 Network Centrality Analysis 3.7 Export Network 4 Notes References Chapter 33: Search, Retrieve, Visualize, and Analyze Protein-Protein Interactions from Multiple Databases: A Guide for Experim... 1 Introduction 2 Materials 3 Methods 3.1 Cytoscape Installation and Launch 3.2 Cytoscape Plugin Installation 3.3 Essential Components of Cytoscape Used in This Protocol 3.4 Import Protein Interactions Using PSICQUIC 3.5 Import Protein Interactions Using the IntAct App 3.6 Import Protein Interactions Using stringApp 3.7 Merging Networks Imported from PSICQUIC, IntAct App, and stringApp 4 Notes References Chapter 34: Centrality Analysis of Protein-Protein Interaction Networks Using R 1 Introduction 2 Materials 3 Methods 3.1 Installing R and Network Analysis Packages 3.2 Loading Network Analysis Packages in R 3.3 Reading Network from a File 3.4 Computing Centrality Indices 3.5 Exporting Centrality Indices to a File 4 Notes References Chapter 35: Protein-Protein Interaction Network Analysis Using NetworkX 1 Introduction 2 Materials 2.1 Installation of NetworkX (Windows, Linux) 2.2 Install NetworkX on Linux 2.3 Use NetworkX in Python (Jupyter Notebook) 3 Methods 3.1 Use Network Datasets in NetworkX for Protein-Protein Interaction Analysis 3.2 Import Required Python Libraries 3.2.1 Load the Data 3.2.2 Make the Network Graph from AI-1MAIN Dataset 3.2.3 Network Centrality Analysis 3.2.4 Exporting the Network 4 Notes References Chapter 36: Building Protein-Protein Interaction Graph Database Using Neo4j 1 Introduction 2 Materials 2.1 Plant Interactome Dataset 2.2 GO Annotation (TAIR) 2.3 Hardware and Software 3 Methods 3.1 Preprocessing of PPI and Annotation Data 3.2 Neo4j Download and Installation Setup 3.3 Import Network and Annotation Data in Neo4j Database 3.4 Graph Centrality Analysis 3.5 Export Analyzed Data 4 Notes References Index