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Protein Termini Part A.

Protein termini represent a major route to protein regulation.From the moment the very first amino acid of a polypeptide chain exits the ribosome there is potential for steering from the cellular environment.

Bibliographic Details
Main Author: Arnesen, Thomas
Corporate Author: ScienceDirect (Online service)
Other Authors: Thomas Arnesen
Format: eBook
Language:English
Published: Chantilly : Elsevier Science & Technology, 2025.
Edition:1st ed.
Series:Methods in Enzymology Series.
Subjects:
Enzymology.
Proteins > Synthesis.
Proteins.
Enzymologie.
Protéines > Synthèse.
Protéines.
protein.
Electronic books.
Online Access:Connect to the full text of this electronic book
Table of Contents:
  • Front Cover
  • Series Page
  • Methods in Enzymology
  • Copyright
  • Contents
  • Contributors
  • Chapter One: Expression and purification of methionine aminopeptidases and N-terminal acetyltransferases
  • 1 Introduction
  • 2 Before you begin
  • 2.1 Buffers
  • 3 Key resources table
  • 4 Materials and equipment
  • 4.1 Materials
  • 4.2 Equipment
  • 4.3 Reagents
  • 5 Step-by-step method details
  • 5.1 Expression of MetAPs and NATs
  • 5.2 Purification of HsMetAP1 and HsMetAP2
  • 6 Expected outcomes
  • 6.1 Purification of HsNatA
  • 7 Expected outcomes
  • 7.1 Purification of HsNatA-HypK
  • 8 Expected outcomes
  • 8.1 Purification of HsNAA50
  • 9 Expected outcomes
  • 10 Optimization and troubleshooting
  • 10.1 The expression did not work
  • 10.2 Protein is not eluting from the column after protease incubation
  • 10.3 MetAP2 shows a degradation pattern on the SDS-gel
  • 10.4 The lysate is not moving through the column
  • References
  • Chapter Two: Purification and activity assays of N-terminal acetyltransferase D
  • 1 Introduction
  • 2 Preparation of recombinant NatD
  • 2.1 Equipment
  • 2.2 Material and buffer recipes
  • 2.3 Transformation
  • 2.4 Expression
  • 2.5 Purification
  • 2.5.1 Preparation of cell lysate
  • 2.5.2 Enrichment of His-TEV-NatD through Ni-NTA column
  • 2.5.3 Cleavage of His-TEV tag
  • 2.6 Expected outcomes
  • 2.7 Notes
  • 3 Continuous fluorescence acetyltransferase assay
  • 3.1 Equipment
  • 3.2 Material and buffer recipes
  • 3.3 Fluorescence assay procedures
  • 3.3.1 Optimization of ThioGlo4 concentration
  • 3.3.2 Standard curve of fluorescence intensity versus [CoASH]
  • 3.3.3 Determine the optimal [enzyme]
  • 3.3.4 Peptide Km study
  • 3.3.5 AcCoA Km study
  • 3.3.6 IC50 study
  • 3.4 Data analysis
  • 3.4.1 AcCoA standard curve
  • 3.4.2 Km of peptide or AcCoA
  • 3.4.3 IC50 study
  • 3.5 Notes
  • 4 MALDI-MS acetyltransferase assay.
  • 4.1 Equipment
  • 4.2 Material and buffer recipes
  • 4.3 MALDI-MS assay procedures
  • 4.3.1 Peptide Km study
  • 4.3.2 IC50 study
  • 4.4 Data analysis
  • 4.4.1 Peptide Km study
  • 4.4.2 IC50 study
  • 4.5 Notes
  • 5 Summary and conclusions
  • Acknowledgment
  • References
  • Chapter Three: A fluorescent CPM-based in vitro acetylation assay: A tool for assessing N-terminal acetyltransferase activity and profiling compound activity
  • 1 Introduction
  • 2 Method overview
  • 3 Equipment and reagent preparation
  • 3.1 Equipment for N-terminal acetylation assay
  • 3.1.1 Microplates
  • 3.1.2 Plate readers
  • 3.2 Preparation of reagents for the N-terminal acetylation assay
  • 3.2.1 Expression and purification of N-terminal acetyltransferases A and B
  • 3.2.1.1 NatA purification
  • 3.2.1.2 NatB purification
  • 3.2.1.3 Notes
  • 3.2.2 Preparation of reagents for N-terminal acetylation assay
  • 3.2.2.1 Ac-CoA
  • 3.2.2.2 CoA
  • 3.2.2.3 Peptides
  • 3.2.2.4 CPM
  • 3.2.2.5 Reaction buffer
  • 3.2.2.6 Bisubstrate inhibitor (CoA-SASEA)
  • 3.2.2.7 Notes
  • 4 Readout reaction CPM-CoA
  • 4.1 CoA-dependent CPM signal and its temporal stability
  • 4.1.1 Procedure
  • 4.2 Linear relationship between CoA concentration and CPM fluorescence
  • 4.2.1 Procedure
  • 4.3 Notes
  • 5 Establishing assay conditions and Michaelis-Menten kinetics
  • 5.1 Optimization of NatA and NatB concentrations
  • 5.1.1 Procedure
  • 5.2 Time course with fixed NatA concentration
  • 5.2.1 Procedure
  • 5.3 Reaction linearity at selected substrate concentrations
  • 5.3.1 Procedure
  • 5.4 Michaelis-Menten kinetics: Km and kcat for peptide and Ac-CoA
  • 5.4.1 Procedure
  • 5.5 Notes
  • 6 Miniaturization of assay to 384-well format
  • 6.1 Procedure
  • 6.2 Notes
  • 7 Test of NatA inhibition (bisubstrate inhibitor)
  • 7.1 Procedure
  • 7.2 Notes
  • 8 Compound screening
  • 8.1 Equipment
  • 8.2 Reagents
  • 8.3 Procedure.
  • 8.4 Notes
  • 9 Conclusions and outlook
  • Acknowledgments
  • References
  • Chapter Four: In vitro acetyltransferase activity assays for N-terminal acetyltransferases
  • 1 Introduction
  • 2 Overview of NAT activity assays
  • 3 NAT enzyme preparation
  • 4 Activity assays
  • 4.1 14C Radioactive acetyltransferase assay
  • 4.1.1 Materials and instrumentation
  • 4.1.2 Reaction conditions
  • 4.2 Fluorescence assay
  • 4.2.1 Materials and instrumentation
  • 4.2.2 Remove free CoA in AcCoA
  • 4.2.3 Reaction conditions
  • 4.2.4 Kinetic analysis of Michaelis-Menten constants (Km)
  • 4.3 ACSS2 coupled luminescence assay
  • 4.3.1 ACSS2 purification
  • 4.3.2 Materials and instrumentation
  • 4.3.3 Reaction conditions
  • 4.4 Comparison between assays
  • 5 Prospects and conclusions
  • Acknowledgments
  • References
  • Chapter Five: Parallel reaction monitoring reveals N-terminal acetylation of plastid precursor proteins
  • 1 Introduction
  • 2 Experimental design
  • 3 General considerations
  • 4 Protoplast protein import assay
  • 4.1 Equipment, materials and buffer recipes
  • 4.1.1 Equipment
  • 4.1.2 Materials
  • 4.1.3 Buffer components
  • 4.1.4 Plasmid construction
  • 4.2 Procedures
  • 4.2.1 Plant growth
  • 4.2.2 Protoplast isolation, transformation and protein extraction
  • 4.2.3 SDS-PAGE and Immunoblot analysis
  • 5 LC-MS/MS and N-terminal peptide identification via Skyline
  • 5.1 Equipment, materials, and buffer recipes
  • 5.1.1 Equipment
  • 5.1.2 Material
  • 5.1.3 Buffer components
  • 5.2 Procedures
  • 5.2.1 Sample preparation and tryptic in-gel digest
  • 5.2.2 Targeted LC-MS/MS
  • 5.2.3 Data analysis
  • 6 Conclusion
  • Acknowledgments
  • References
  • Chapter Six: N-terminal acetylation-specific antibodies: Specificity determination by mass spectrometry and utilization in in vitro acetylation assays
  • 1 Introduction
  • 2 Methods.
  • 3.4.2 SPOT array
  • 4 Development of anti-pan-N-fMet-specific antibody using a mixed antigen
  • 4.1 Antigen design
  • 4.2 Antibody production
  • 5 Analysis of protein Nt-formylation
  • 5.1 Detection in Escherichia coli
  • 5.1.1 Equipment, materials, and buffer recipes
  • 5.1.2 Cell growth and lysis
  • 5.1.3 Immunoblotting with pan-fMet-specific antibodies
  • 5.2 Detection in Salmonella Typhimurium
  • 5.2.1 Equipment, materials, and buffer recipes
  • 5.2.2 Cell growth
  • 5.2.3 Cell lysis
  • 5.2.4 Immunoblotting with pan-fMet antibodies
  • 5.3 Detection in yeast mitochondria
  • 5.3.1 Equipment, materials, and buffer recipes
  • 5.3.2 Cell culture
  • 5.3.3 Mitochondria fractionation and lysis
  • 5.3.4 Immunoblotting of fMet-proteins with pan-fMet-specific antibodies
  • 5.4 Detection in human cells
  • 5.4.1 Equipment, materials, and buffer recipes
  • 5.4.2 Cell culture
  • 5.4.3 Cell lysis
  • 5.4.4 Immunoblotting with pan-fMet antibodies
  • 5.5 Detection of synthetic peptides
  • 5.5.1 Equipment, materials, and buffer recipes
  • 5.5.2 Peptide conjugation
  • 5.5.3 ELISA with pan-fMet-specific antibodies
  • 6 Concluding remarks
  • Acknowledgments
  • Disclosure statement
  • References
  • Chapter Nine: Chemical proteomic approaches to investigate N-myristoylation
  • 1 Introduction
  • 2 General proteomic considerations
  • 3 Metabolic labelling protocol
  • 3.1 Prior considerations
  • 3.2 Cell treatment
  • 3.2.1 Equipment, buffers and reagents
  • 3.2.2 Procedure
  • 3.3 Bio-orthogonal click reaction and tag ligation
  • 3.3.1 Equipment, buffers and reagents
  • 3.3.2 Procedure
  • 3.4 Protein enrichment
  • 3.4.1 Equipment, buffers and reagents
  • 3.4.2 Procedure
  • 3.5 Cysteine alkylation and protein digest
  • 3.5.1 Equipment, buffers and reagents
  • 3.5.2 Procedure
  • 3.6 Peptide preparation and LC-MS/MS analysis
  • 3.6.1 Equipment, buffers and reagents.