LysargiNase and Chemical Derivatization Based Strategy for Facilitating In-Depth Profiling of C-Terminome.
Anal Chem
; 91(22): 14522-14529, 2019 11 19.
Article
em En
| MEDLINE
| ID: mdl-31634432
ABSTRACT
Global identification of protein C-termini is highly challenging due to their low abundance in conventional shotgun proteomics. Several enrichment strategies have been developed to facilitate the detection of C-terminal peptides. One major issue of previous approaches is the limited C-terminome coverage. Herein, we integrated LysargiNase digestion, chemical acetylation on neo-N-terminus, and a-ion-aided peptide matching into poly(allylamine)-based C-terminomics (termed as LAACTer). In this strategy, we leveraged LysargiNase, a protease with cleavage specificity N-terminal to Lys and Arg residues, to cover previously unidentifiable C-terminome and employed chemical acetylation and a-ion-aided peptide matching to efficiently boost peptide identifications. Triplicates of LAACTer identified a total of 834 C-termini from proteome of 293T cell, which expanded the coverage by 164% (643 more unique C-termini) compared with the parallel experiments using the original workflow. Compared with the largest human C-terminome data sets (containing 800-900 C-termini), LAACTer not only achieved comparable profiling depth but also yielded 465 previously unidentified C-termini. In a SILAC (stable isotope labeling with amino acids in cell culture)-based quantitative study for identification of GluC-cleaved products, LAACTer quantified 300% more C-terminal peptides than the original workflow. Using LAACTer and the original workflow, we performed global analysis for the C-terminal sequences of 293T cell. The original and processed C-termini displayed distinct sequence patterns, implying the "C-end rules" that regulates protein stability could be more complex than just amino acid motifs. In conclusion, we reason LAACTer could be a powerful proteomic tool for in-depth C-terminomics and would benefit better functional understanding of protein C-termini.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Proteoma
/
Metaloproteases
/
Domínios Proteicos
Limite:
Humans
Idioma:
En
Ano de publicação:
2019
Tipo de documento:
Article