Protein Arginylation: Methods and Protocols

Preface
Contents
Contributors
Chapter 1: Protein Arginylation: Milestones of Discovery
1 Early Milestones in Protein Arginylation Studies
References
Chapter 2: Recollection of How We Came Across the Protein Modification with Amino Acids by Aminoacyl tRNA-Protein Transferase
References
Chapter 3: Arginyltransferase: A Personal and Historical Perspective
References
Chapter 4: Arginylation in a Partially Purified Fraction of 150 k xg Supernatants of Axoplasm and Injured Vertebrate Nerves
1 Introduction
2 Materials
2.1 Tissues
2.2 Sample Preparation
2.3 Reactions
3 Methods
3.1 Preparation of Squid Axoplasm
3.2 Preparation of Rat Sciatic Nerves
3.3 Preparation of Rat Optic Nerves
3.4 Preparation of the Active Subcellular Fractions
3.5 Fractionation by Sephacryl S-200 Chromatography
3.6 Posttranslational Incorporation of Amino Acids into Proteins
3.7 Determination of Protein Arginylation
4 Notes
References
Chapter 5: Preparation of ATE1 Enzyme from Native Mammalian Tissues
1 Introduction
2 Materials
3 Methods
3.1 Preparation of Tissue Extract
3.2 Ammonium Sulfate Fractionation
3.3 Precipitation at pH 5
3.4 Column Chromatography
4 Notes
References
Chapter 6: Correlated Measurement of Endogenous ATE1 Activity on Native Acceptor Proteins in Tissues and Cultured Cells to Det...
1 Introduction
2 Materials
3 Methods
3.1 Preparation of Chromatin (Arg Acceptor) and Post-microsomal Supernatant (Source of the ATE1 Enzyme) from Rat Tissues for R...
3.2 Preparation of Chromatin and Post-microsomal Supernatant from Cultured Cells for Cell Aging Studies
3.3 Preparation of Arginyl-tRNA Transferase (ATE1), tRNA, and Arginyl-tRNA
3.4 Assay of Arginyl-tRNA Synthetase and Arginyl-tRNA Transferase Activity Using an ATP Regenerating System
3.5 Determination of Cell Population Doubling Time
3.6 Transformation of Cells with SV40 Virus
4 Notes
References
Chapter 7: Assaying the Posttranslational Arginylation of Proteins in Cultured Cells
1 Introduction
2 Materials
2.1 Cell Culture
2.2 Buffers and Solutions
2.3 Reagents
2.4 Equipment
2.5 Chemicals
3 Methods
3.1 Incorporation of [14C]Arg into Proteins from Cultured Cells
3.2 Evaluation of [14C]Arg Incorporation to the N-Terminal Position of Proteins
4 Notes
References
Chapter 8: Assaying ATE1 Activity in Yeast by β-Gal Degradation
1 Introduction
2 Materials
3 Methods
3.1 Preparation of Yeast Competent Cells
3.2 Yeast Transformation
3.3 Color Overlay Assay for Beta-Gal Activity Using X-Gal
3.4 Liquid Assay for Beta-Gal Activity Using ONPG
4 Notes
References
Chapter 9: Assaying Arginylation Activity in Cell Lysates Using a Fluorescent Reporter
1 Introduction
2 Materials
3 Methods
3.1 Cloning of DD-beta15-GFP Arginylation Reporter
3.2 Production of DD-beta15-GFP Arginylation Reporter in Yeast and Its Purification Using GFP-TRAPS Nanobody Conjugated with M...
3.3 In-Lysate Arginylation Assay in Yeast
3.4 In-Lysate Arginylation Assay in Mammalian Cells
3.5 Western Blot Analysis
4 Notes
References
Chapter 10: Assaying Intracellular Arginylation Activity Using a Fluorescent Reporter
1 Introduction
2 Materials
3 Methods
4 Notes
References
Chapter 11: Bacterial Expression and Purification of Recombinant Arginyltransferase (ATE1) and Arg-tRNA Synthetase (RRS) for A...
1 Introduction
2 Materials
3 Methods
3.1 Expression and Purification of ATE1
3.2 Expression and Purification of RRS
4 Notes
References
Chapter 12: Preparation of tRNAArg for Arginylation Assay by In Vitro Transcription
1 Introduction
2 Materials
3 Methods
3.1 Transcription Template Design
3.2 Preparation of the Transcription Template from tDNA Oligonucleotides
3.3 In Vitro Transcription
4 Notes
References
Chapter 13: Preparation of an Enriched tRNAArg Fraction for Arginylation by Expression in E. coli
1 Introduction
2 Materials
2.1 Materials for Bacterial Cell Transformation and Growth
2.2 Materials for tRNA Isolation
3 Methods
3.1 tRNA Expression in Bacterial Cells
3.2 tRNA Isolation
4 Notes
References
Chapter 14: Enzymatic Aminoacylation of tRNAArg Using Recombinant Arg-tRNA Synthetase
1 Introduction
2 Materials
3 Methods
4 Notes
References
Chapter 15: Assaying ATE1 Activity In Vitro
1 Introduction
2 Materials
3 Methods
3.1 Individual Format Assay Using a Protein ATE1 Substrate
3.2 Individual Format Assay Using a Peptide ATE1 Substrate
4 Notes
References
Chapter 16: High-Throughput Arginylation Assay in Microplate Format
1 Introduction
2 Materials
3 Methods
4 Notes
References
Chapter 17: Assaying for Arginyltransferase Activity and Specificity by Peptide Arrays
1 Introduction
2 Materials
3 Methods
4 Notes
References
Chapter 18: Assay of Arginyltransferase Activity by a Fluorescent HPLC Method
1 Introduction
2 Materials
2.1 Materials for Chemical Syntheses of Fluorescent Substrate and Product
2.2 Materials for HPLC Analysis
2.3 Materials for the Assay of Arginyltransferase Activity
3 Methods
3.1 Chemical Syntheses of Fluorescent Substrate and Product
3.1.1 Synthesis of 4-Dansylamidobutylamine (4DNS)
3.1.2 Synthesis of N-Aspartyl-4-Dansylamidobutylamide (Asp-4DNS)
3.1.3 Synthesis of N-Arginylaspartyl-4-Dansylamidobutylamide (ArgAsp-4DNS)
3.2 HPLC Analysis
3.3 Assay of Arginyltransferase Activity
3.3.1 Preparation of Arginyl-tRNA Synthetase from E. coli (See Note 8)
3.3.2 Preparation of Arginyltransferase from Hog Kidney as a Standard Enzyme
3.3.3 Preparation of Crude Supernatants from Animal Tissues
3.3.4 Assay of Arginyltransferase Activity in Crude Supernatants
4 Notes
References
Chapter 19: Identification of Arginylated Proteins by Mass Spectrometry
1 Introduction
2 Materials (See Note 1)
3 Methods
3.1 Sample Preparation and Mass Spectrometry
3.2 Identification of Arginylated Peptides
4 Notes
References
Chapter 20: Analysis of Arginylated Peptides by Subtractive Edman Degradation
1 Introduction
2 Materials
3 Methods
4 Note
References
Chapter 21: Transferase-Mediated Labeling of Protein N-Termini with Click Chemistry Handles
1 Introduction
2 Materials
2.1 Enzymes and Plasmids
2.2 Buffers for Protein Expression
2.3 Materials for Protein Expression
2.4 Materials for Azf-a Synthesis and Chemoenzymatic Transfer Reactions
2.5 Materials for Fully Enzymatic Transfer Reactions
2.6 Materials for HPLC Transfer Assays
2.7 Materials for Transfer Reactions in Cell Lysates
2.8 Materials for Click Chemistry Labeling of Azide-Derived Proteins
3 Methods
3.1 E. Coli Leucyl Phenylalanyl Aminoacyl Transferase (AaT) Expression and Purification
3.2 Azidophenylalanine Synthetase (AzfRS) Expression and Purification
3.3 Mutant Methionyl Synthetase (Met*RS) Expression and Purification
3.4 Aminoacyl Adenosine Donor Synthesis
3.5 Chemoenzymatic Peptide Transfer Assay
3.6 Fully Enzymatic Peptide Transfer Assay
3.7 Chemoenzymatic AaT Transfer to Proteins in Cell Lysates
3.8 Fully Enzymatic AaT Transfer to Proteins in Cell Lysates
3.9 Copper-Catalyzed Click Labeling
3.10 Strain-Promoted Click Labeling
4 Notes
References
Chapter 22: Synthesis of Peptides and Proteins with Site-Specific Glutamate Arginylation
1 Introduction
2 Materials
2.1 Arginylated Glutamate Monomer Synthesis
2.2 Peptide Synthesis (See Note 1)
2.3 Plasmids and Cloning
2.4 Buffers for Protein Expression
2.5 Protein Expression and Labeling
2.6 Native Chemical Ligation (NCL)
3 Methods
3.1 Synthesis of the Fmoc-Glu(Arg(Pdf)-OtBu)-OH
3.2 Peptide Synthesis
3.2.1 Peptide Synthesis for Site-Specific EArg Antibody Generation (Anti-Arginylated αS)
3.2.2 Peptide Library Synthesis for Pan-EArg Antibody Generation
3.2.3 Peptide Synthesis for Cross-Linking and Pull-Down Applications
3.2.4 Peptide Synthesis for Arginylated Protein Semi-Synthesis
3.3 Protein Expression for Arginylated Protein Semi-Synthesis
3.3.1 Production of Plasmids Coding αS Fragment-Intein Constructs
3.3.2 Production of N-Terminal Protein Fragment
3.3.3 Production of C-Terminal Protein Fragment (no Labeling)
3.3.4 Production of Fluorescently Labeled C-Terminal Protein Fragment
3.4 Native Chemical Ligation for Arginylated Protein Semi-Synthesis
3.4.1 NCL 1: Ligation Between αS1-76-Thioester and αS77-90- V*77EArg83-Hydrazide
3.4.2 NCL 2: Ligation Between αS1-90-V*77EArg83-Hydrazide and αS91-140-C91/αS91-140-C91π488114 Followed by Desulfurization, Us...
3.4.3 NCL 2: Ligation between αS1-90-V*77EArg83-Hydrazide and αS91-140-C91/ αS91-140-C91π488114 Followed by Desulfurization, U...
4 Notes
References
Chapter 23: Reconstitution of the Arginyltransferase (ATE1) Iron-Sulfur Cluster
1 Introduction
2 Materials
2.1 Chemical Reconstitution
2.2 Ferrozine Assay
3 Methods
3.1 Bringing Materials into Anaerobic Chamber
3.2 Chemical Reconstitution of Protein
3.3 Removal of Excess Iron Sulfide
3.4 Electronic Absorption Spectroscopy and the Ferrozine Assay to Assess Cluster Loading
4 Notes
References
Chapter 24: N-Terminal Arginylation Pull-down Analysis Using the R-Catcher Tool
1 Introduction
2 Materials
3 Methods
3.1 Expression and Purification of R-Catcher
3.2 Immobilization of R-Catcher on Resin
3.3 Preparing Cell Lysates for R-Catcher Pull-down
3.3.1 Induction and Analysis of Endogenous Arginylated Proteins
3.3.2 Preparation and Analysis of Exogenous Arginylated Proteins
3.4 R-Catcher Pull-down Assay with Peptide Competition
3.5 Analysis of R-Catcher Pull-down Proteins
3.5.1 Western Blotting
3.5.2 Silver Staining
3.6 R-Catcher Pull-down Assay for LC/MS Analysis
3.6.1 Resin Preparation
3.6.2 Cell Lysate Preparation
3.6.3 R-Catcher Pull-down of Arginylated Proteins
4 Notes
References
Chapter 25: Identification of Protein Arginylation by Encapsulated N-Terminal Peptide Enrichment Method
1 Introduction
2 Materials
3 Methods
3.1 Cell Lysis and Thiol Blocking
3.2 Labeling of Primary Amines and Digestion to Peptides (for 25-200 μg)
3.3 Negative Selection of N-terminal Peptides (for 25-200 μg)
3.4 Labeling of Primary Amines and Digestion to Peptides (for 200 μg-5 mg)
3.5 Negative Selection of N-Terminal Peptides (for 200 μg-5 mg)
3.6 bRPLC Fractionation (Only for 200 μg-5 mg)
3.7 LC-MS/MS Analysis
3.8 N-Terminal Arginylome Data Analysis
4 Notes
References
Chapter 26: Monitoring the Activation of Selective Autophagy via N-Terminal Arginylation
1 Introduction
2 Materials
2.1 In Vitro p62/SQSTM1 Oligomerization Assay
2.2 Punctate Formation and Co-localization Analysis
2.3 Detergent-Insoluble/Soluble Fractionation-Based Autophagy Flux
3 Methods
3.1 In Vitro p62/SQSTM1 Oligomerization Assay
3.2 Punctate Formation and Co-localization Analysis
3.3 Detergent-Insoluble/Soluble Fractionation-Based Autophagy Flux
4 Notes
References
Chapter 27: Analyzing the Interaction of Arginylated Proteins and Nt-Arg-Mimicking Chemical Compounds to N-Recognins
1 Introduction
2 Materials
2.1 Pull-Down Assay
2.1.1 Peptide Conjugation
2.1.2 Preparation of Cell Lysate
2.1.3 Pull-Down Assay
2.2 MST (MicroScale Thermophoresis)
3 Methods
3.1 Pull-Down Assay
3.1.1 Peptide Conjugation
3.1.2 Preparation of Cell Lysate
3.1.3 Pull-Down Assay
3.2 MST (MicroScale Thermophoresis)
3.2.1 Peptide Conjugation
3.2.2 Protein Buffer Exchange
3.2.3 Protein Staining
3.2.4 MST
4 Notes
References
Chapter 28: Synthesis of Stably Charged Arg-tRNAArg for Structural Analysis
1 Introduction
2 Materials
3 Methods
3.1 Bacterial Cell Transformation and Growth
3.2 Isolation of the Total tRNA Pool and Deacylation
3.3 3′-Amino Tailing of the tRNA Pool
3.4 Affinity Purification of tRNAArg(ICG) from the 3′-Tailed Total tRNA Pool
3.5 Removal of the Capture Oligonucleotide
3.6 Stable Charging of tRNAArg(ICG)
3.7 Calculation of the Stable Charging Efficiency
3.8 Perform a Preparative Charging Reaction
4 Notes
References
Chapter 29: A High-Throughput Colorimetric Microplate Assay for Determination of Plasma Arginase Activity
1 Introduction
2 Materials
2.1 Plasma Isolation by Density Gradient Centrifugation
2.2 Colorimetric Microplate Assay
2.3 Analysis
3 Methods
3.1 Plasma Isolation by Density Gradient Centrifugation
3.2 Standard Curve
3.3 Serial Dilution of Plasma
3.4 Colorimetric Assay
3.5 Analysis
4 Notes
References
Chapter 30: Development of New Tools for the Studies of Protein Arginylation
1 ``Pan-Arginylation´´ Antibodies
2 Antibodies to Ate1
3 Peptide Antibodies to Individual Arginylated Proteins
4 Other Approaches to Arginylation Detection
References
Index