Cell-Wide Identification of Metabolite-Protein Interactions (Methods in Molecular Biology, 2554)

Description

This thorough volume explores protocols of proteome- and metabolome-wide strategies for the identification of protein-small molecule complexes in different organisms, in order to shed light on these important regulatory interactions. Experimental and computational strategies to characterize protein-metabolite interactions are discussed, and recent advances in enabling technologies are featured as well. Written for the highly successful Methods in Molecular Biology series, chapters include the kind of detail and expert implementation advice to ensure success in future research.  Authoritative and practical, Cell-Wide Identification of Metabolite-Protein Interactions will aid researchers seeking a better understanding of the mechanisms of signal transduction occurring in the cell and assessing the effect of complex formation on cell physiology. Preface Contents Contributors Chapter 1: Protein-Metabolite Interactions Shape Cellular Metabolism and Physiology 1 Introduction 2 PMIs Regulating Metabolic Enzymes 3 PMIs Regulating Transcription 4 PMIs in Disease Development and Treatment 5 Concluding Remarks References Chapter 2: Affinity Purification Protocol Starting with a Small Molecule as Bait 1 Introduction 2 Materials 2.1 Plant Material and Native Lysate 2.2 Affinity Purification 2.3 SDS Protein Gel 2.4 Protein Preparation for Proteomics 2.5 Proteomic Run 3 Methods 3.1 Plant Growth and Native Arabidopsis Lysate Preparation (See Note 1) 3.2 Affinity Purification (See Note 3) 3.3 SDS Protein Gel (See Note 4) 3.4 Proteomics: Sample Preparation (See Note 5) 3.4.1 Enzymatic Digestion 3.4.2 Desalting (See Note 6) 3.5 LC/MS-MS Protemics and Data Analysis (See Note 7) 4 Notes References Chapter 3: Cellular Thermal Shift Assay for the Detection of Small Molecule-Target Interactions in Arabidopsis Cells 1 Introduction 2 Materials 2.1 CETSA 2.2 Immunoblot 2.3 TMT-Based Mass Spectrometry 3 Methods 3.1 CETSA on Cells 3.2 CETSA on Seedlings 3.3 Immunoblots 3.3.1 Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) 3.3.2 Blot Transfer 3.3.3 Immunoblot 3.4 Protein Reduction, Alkylation, and Digestion and TMT Labeling 4 Notes References Chapter 4: Solvent-Induced Protein Precipitation for Drug Target Discovery 1 Introduction 2 Materials 2.1 Solvent-Induced Precipitation 2.2 Mass Spectrometry Analysis 2.3 Western Blotting 3 Methods 3.1 Sample Preparation by SIP for Stability Profiling 3.2 Identification of Potential Drug Targets 3.3 Validation of Potential Drug Targets 4 Notes References Chapter 5: Identification of Plant Protein-Metabolite Interactions by Limited Proteolysis-Coupled Mass Spectrometry (LiP-MS) 1 Introduction 2 Materials 2.1 Sample Preparation 2.2 Mass Spectrometry Data Acquisition 2.3 Data Analysis 3 Methods 3.1 Sample Preparation 3.2 Mass Spectrometry Data Acquisition 3.3 DDA Data Analysis 3.4 DIA Data Analysis 3.4.1 Library Generation for DIA Analysis 3.4.2 DIA Analysis Using OpenSwathWorkflow 3.4.3 Statistical Analysis with DEP 4 Notes References Chapter 6: Limited Proteolysis-Mass Spectrometry to Identify Metabolite-Protein Interactions 1 Introduction 2 Materials 2.1 Cell Lysis Under Native Conditions 2.2 Preparation of Stock Solutions of Metabolites 2.3 Limited Proteolysis Under Native Conditions 2.4 Sample Preparation Prior to MS Analysis 2.5 LC-MS/MS Data Acquisition 2.6 Software 3 Methods 3.1 Cell Lysis Under Native Conditions 3.2 Preparation of Stock Solutions of Metabolites 3.3 Limited Proteolysis 3.4 Sample Preparation Prior to MS Analysis 3.5 LC-MS/MS Analysis 3.6 Library Generation in Spectronaut 3.7 Performing a DIA Library-Based Analysis in Spectronaut 3.8 Post-analysis Processing 3.9 LiP-Quant Analysis 3.10 Running LiP-Quant Analysis 3.10.1 Installation of R, Required Packages, and Adjustment of Settings 3.10.2 Machine Learning-Based Determination of Adjusted Score Weights 3.10.3 LiP-Quant Analysis of New Targets 4 Notes References Chapter 7: Proteome Integral Solubility Alteration (PISA) for High-Throughput Ligand Target Deconvolution with Increased Stati... 1 Introduction 2 Materials 2.1 Cell Culture and Ligand Treatment 2.2 PISA Treatment (Thermal Treatment, Cell Lysis, and Soluble Fraction Separation) 2.3 Soluble Protein Fraction Processing 2.4 Peptide Separation, Mass Spectrometry, and Proteomics Data Analysis 3 Methods 3.1 Cell Culture and Ligand Treatment 3.2 PISA Treatment (Thermal Treatment, Cell Lysis, and Soluble Fraction Separation) 3.2.1 Protein Extraction of Carrier Proteome Sample 3.2.2 Preparation of PISA-Treated Samples 3.3 Soluble Protein Fraction Processing 3.4 Peptide Separation, Mass Spectrometry, and Proteomics Data Analysis 4 Notes References Chapter 8: Parallel Analysis of Protein-Protein and Protein-Metabolite Complexes Using a Single-Step Affinity Purification 1 Introduction 2 Materials 2.1 Plant Cell Culture 2.2 Tap 2.3 Immunodetection of Protein Bait 2.4 Metabolite Extraction 2.5 Proteomics: Preparation 2.6 Proteomic Analysis 2.7 Metabolomic Analysis 3 Methods 3.1 Plant Cell Culture (See Note 1) 3.2 Tandem Affinity Purification (Adapted from) (Fig. 2) 3.3 Immunodetection of Protein Bait 3.3.1 SDS-Page 3.3.2 Protein Gel Blotting 3.3.3 Immunodetection 3.4 Metabolite and Protein Extraction (Adapted from) (See Note 4) 3.5 Proteomics: Sample Preparation 3.5.1 Enzymatic Digestion (See Note 5) 3.5.2 Desalting (See Note 6) 3.6 Proteomic Analysis (See Note 7) 3.7 Sample Preparation 3.7.1 Sample Separation and Measurement Using Liquid Chromatography (C18 Reversed-Phase Column) Coupled to a Mass Spectrometer 3.7.2 Proteomic Data Analysis 3.8 Metabolomic Analysis of Polar Phase (See Note 7) 3.8.1 Sample Preparation 3.8.2 Metabolomic Analysis Using UPLC (C18 Reversed-Phase Column) Coupled to a Mass Spectrometer 3.8.3 Metabolomic Data Analysis 4 Notes References Chapter 9: Detecting and Characterizing Interactions of Metabolites with Proteins by Saturation Transfer Difference Nuclear Ma... 1 Introduction 2 Materials 2.1 Protein Expression 2.2 NMR Sample Preparation 2.3 NMR Equipment and Software 3 Methods 3.1 Protein Expression 3.2 NMR Sample Preparation 3.3 Acquisition of NMR Spectra 3.3.1 1H NMR Experiments 3.3.2 STD NMR Experiments 3.3.3 STD NMR Control Experiments 3.3.4 NMR Experiment for Ligand Assignment 3.4 Processing of STD NMR Spectra 4 Notes References Chapter 10: PROMIS: Co-fractionation Mass Spectrometry for Analysis of Protein-Metabolite Interactions 1 Introduction 2 Materials 2.1 Plant Material Lysate 2.2 Size-Exclusion Chromatography 2.3 Metabolite and Protein Extraction 2.4 Metabolomic Analysis (Use MS-Grade Reagents) 2.5 Proteomic Analysis (Use MS-Grade Reagents) 3 Methods 3.1 Plant Material Lysate (See Notes 1 and 2) 3.2 Size-Exclusion Chromatography (See Note 4) 3.3 Metabolite and Protein Extraction 3.4 Metabolomic Analysis (See Note 5) 3.4.1 Sample Preparation 3.4.2 LC-MS Measurement 3.4.3 Metabolomic Data Analysis 3.5 Proteomic Analysis 3.5.1 Enzymatic Digestion 3.5.2 Desalting 3.5.3 LC-MS Analysis 3.5.4 Proteomic Data Analysis 3.6 Data Integration 4 Notes References Chapter 11: Metabolomics: Going Deeper, Going Broader, Going Further 1 Introduction 2 Increasing Coverage 2.1 Challenges in Sample Preparation 2.2 Impact of Chromatographic Separation on Coverage 2.3 Impact of MS Settings on Coverage 2.4 Going Deep Versus Going Global 3 Going Smaller 3.1 Miniaturization of Chromatography 3.2 Spatially Resolved Metabolomics 3.3 Other Approaches 4 Identifying More Metabolites with Higher Confidence in MS-Based Untargeted Metabolomics 4.1 MS-Based Metabolite Identification 4.2 Limitations of MS-Based Identification 4.3 Targeted Metabolomics 5 Integration of Metabolomics with Other Omics Data 5.1 Overrepresentation Analyses 5.2 Network-Based Global Analyses 5.3 Systematic Local Analyses 6 Conclusion/Outlook References Chapter 12: Specifics of Metabolite-Protein Interactions and Their Computational Analysis and Prediction 1 Introduction 2 Metabolite-Protein Versus Drug-Protein Interactions: Commonalities and Differences 2.1 Compound Characteristics 2.2 Binding Energetics: Affinities 2.3 Binding Promiscuity 2.4 Multi-Compound Binding: Enzyme-Substrate Complex Formation 3 Predicting and Simulating PMIs 3.1 Catalytic Site Predictions 3.2 Conversion Rate Predictions/Kinetic Parameters 3.3 Simulation of Substrate-Enzyme Encounter 3.4 Prediction of Allosteric Sites and Effects 3.5 Regulation of Metabolism and Compound-Binding Via Posttranslational Modifications 4 Taking a Network-Based View 4.1 Protein-Protein Interactions and Metabolism: Integrative Molecular Network Analysis 4.2 Network-Based Approaches to the Prediction of Metabolite-Protein Interactions 5 Online Tools, Recourses, and Databases 6 Frontiers, Open Questions References Chapter 13: Biophysical Approaches for the Characterization of Protein-Metabolite Interactions 1 Introduction 2 Surface Plasmon Resonance (SPR) 3 Isothermal Titration Calorimetry (ITC) 4 Nuclear Magnetic Resonance (NMR) Spectroscopy 5 Fluorescence Spectroscopy Techniques 5.1 Direct Fluorescence Spectroscopy 5.2 Fluorescence Resonance Energy Transfer (FRET) 5.3 Fluorescence Polarization (FP) 5.4 Fluorescence Correlation Spectroscopy (FCS) 5.5 Microscale Thermophoresis (MST) 5.6 Differential Scanning Fluorimetry (DSF) 6 Vibrational (Fourier Transform Infrared (FTIR) and Raman) Spectroscopy 7 Circular Dichroism (CD) Spectroscopy 8 Solution Scattering Techniques 9 Analytical Ultracentrifugation (AUC) 10 Further Methods 11 Concluding Remarks References Chapter 14: Databases and Tools to Investigate Protein-Metabolite Interactions 1 Introduction 2 The Foundation Techniques of Proteomics and Metabolomics and Useful Databases in Their Application 3 Overview of Techniques to Assess PMIs 3.1 Metabolite-Centric PMI Screening 3.2 Protein-Centric PMI Screening 3.3 Untargeted PMI Screening 4 Databases for PMI 4.1 Manually Curated Databases 4.2 Text Mining and Prediction Databases 4.3 Databases with Negative Interactions 4.4 Databases Containing Large-Scale PMI Screening 5 Other Tools for Data Analysis and Visualization 6 Concluding Remarks References Afterword References Index