Improved Coverage of the N-Terminome by Combining ChaFRADIC with Alternative Proteases.
Methods Mol Biol
; 2718: 99-110, 2023.
Article
em En
| MEDLINE
| ID: mdl-37665456
ABSTRACT
Many proteolytic cleavage events cannot be covered with conventional trypsin-based N-terminomics workflows. These typically involve the derivatization of protein N-termini and Lys residues as an initial step, such that trypsin will cleave C-terminal of arginine but not lysine residues (ArgC-like cleavage). From 20,422 reviewed human protein sequences in Uniprot, 3597 have known N-terminal signal peptides. An in silico ArgC-like digestion of the corresponding 3597 mature protein sequences reveals that-even for these well-known and well-studied proteolytic events-trypsin-based N-terminomics workflows may miss up to 50% of signaling cleavage events as the corresponding neo-N-terminal peptides will have an unfavorable length of <7 (875 peptides) or >30 (911 peptides) amino acids. In this chapter, we provide a protocol that can be applied to all kinds of samples to improve access to this "inaccessible" N-terminome, by making use of the alternative, broad-specificity protease subtilisin for fast and reproducible digestion of proteins.
Palavras-chave
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Peptídeo Hidrolases
/
Aminoácidos
Limite:
Humans
Idioma:
En
Revista:
Methods Mol Biol
Assunto da revista:
BIOLOGIA MOLECULAR
Ano de publicação:
2023
Tipo de documento:
Article
País de afiliação:
Canadá