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.
1. Proteome analysis of puromycin-labeled nascent polypeptides
Koshi Imami
2. HUNTER-DIA: An updated protocol for enrichment and mass spectrometry-based identification of protein N-termini
Pitter F. Huesgen
3. TERMINER - Bioinformatic detection and annotation of proteolytic protein termini in shotgun proteomics data
Oliver Schilling
4. Degronopedia: A practical guide to identifying and targeting protein degrons
Wojciech Pokrzywa
5. Quantitative Insights into Protein Turnover and Ubiquitination with HiBiT and NanoBRET
Wojciech Pokrzywa
6. Development of a flow cytometric method to evaluate the impact of N-terminal sequences on protein stability
Aditya M. Kunjapur
7. Generation and characterization of engineered N-degrons of the N-degron pathway using the ubiquitin-reference technique
Chang Hoon Ji
8. Characterization of the autophagic N-degron pathway and monitoring its chemical modulation for therapeutic development
Yong Tae Kwon
9. Characterization of the E3 ligase KCMF1 as a ZZ/N-recognin of the autophagic Arg/N-degron pathway
Chang Hoon Ji
10. Identification of Ac/N-degron-recognition domain within the MARCHF6 E3 ubiquitin ligase
Cheol-Sang Hwang
11. Affinity purification-mass spectrometry to identify nuclear protein interactions of N-terminal acetyltransferase NAA40
Antonis Kirmizis
12. TurboID technique for proximity labelling of interacting proteins
Greta Jarck
13. Chemical proteomic approaches to investigate S-prenylation
Edward W. Tate
14. Quantitative analysis of C-terminal prenylated protein levels using tandem mass tagging
Mark Distefano
15. Optimizing purification and FP-based binding assays for the E3 ligase FEM1C
Rong Huang
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