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. This volume of Methods in Enzymology Modifications and Targeting of Protein Termini focuses on Protein N-termini and C-termini and their modifications which include acetylation, arginylation, myristoylation and oxidation. Also, the impact of terminal modifications is covered, in particular the impact on protein turnover and the ubiquitin E3 ligases which specifically recognize protein N-termini (N-degrons) and C-termini (C-degrons). In addition to the detailed methods and laboratory protocols, the chapters include informative overviews and reviews of the different subfields.
Table of Contents
1. In vitro acetyltransferase assays for NAT enzymesRonen Marmorstein
2. A fluorescent CPM-based in vitro acetylation assay: a tool for assessing N-Terminal acetyltransferase activity and profiling compound activity
Tanja Bange
3. Methods for Purification and Activity Analysis of N-Terminal Acetyltransferases
Rong Huang
4. N-terminal acetylation of plastid precursor proteins
Sacha Baginsky
5. Utilizing N-terminal acetylation specific antibodies
Henriette Aksnes
6. Functional assessment of N-terminal acetyltransferase variants
Nina McTiernan
7. Affinity purification-mass spectrometry to identify nuclear protein interactions of N-terminal acetyltransferase NAA40
Antonis Kirmizis
8. A pan-N-formylmethionine-specific antibody as a tool to for analyzing N??-terminal formylation
Cheol-Sang Hwang
9. Chemical proteomic approaches to investigate N-myristoylation
Edward W. Tate
10. Analysis of the oxidation state of N-terminal methionines
Sina Ghaemmaghami
11. A biophysical approach to studying N-terminal cysteine oxidase substrate preferences
Mark White
12. Recombinant expression, purification, and characterization of human ATE1 arginyltransferase
Yi Zhang
13. In-bacteria arginylation assay
Yi Zhang
14. Identification of arginylated N-termini with specific antibodies
Jasper Eising
15. Characterization of the autophagic N-degron pathway and monitoring its chemical modulation for therapeutic development
Yong Tae Kwon
16. Developing a flow cytometric method to evaluate the stability of protein N-termini
Aditya M. Kunjapur
17. Degronopedia: A practical guide to identifying and targeting protein degrons
Wojciech Pokrzywa
18. Generation and usage of ubiquitin-reference technique (URT) plasmids to validate N-degrons
Chang Hoon Ji
19. Quantitative Insights into Protein Turnover and Ubiquitination with HiBiT and NanoBRET
Wojciech Pokrzywa
20. Characterization of the autophagy E3 ligase/N-recognin KCMF1
Chang Hoon Ji
21. Identification of Ac/N-degron-recognition domain within the MARCHF6 E3 ubiquitin ligase
Cheol-Sang Hwang
22. TurboID technique for proximity labelling of interacting proteins
Greta Jarck
23. Proteome analysis of nascent polypeptide chains using puromycin
Koshi Imami
24. HUNTER-DIA: An updated protocol for enrichment and mass spectrometry-based identification of protein N-termini
Pitter F. Huesgen
25. TERMINER Bioinformatic detection and annotation of proteolytic protein termini in shotgun proteomics data
Oliver Schilling
26. Chemical proteomic approaches to investigate S-prenylation
Edward W. Tate
27. Quantitative analysis of C-terminal prenylated protein levels using tandem mass tagging
Mark Distefano
28. Optimizing purification and FP-based binding assays for the E3 ligase FEM1C
Rong Huang
