Development of a novel cross-linking strategy for fast and accurate identification of cross-linked peptides of protein complexes.
Mol Cell Proteomics
; 10(1): M110.002212, 2011 Jan.
Article
em En
| MEDLINE
| ID: mdl-20736410
ABSTRACT
Knowledge of elaborate structures of protein complexes is fundamental for understanding their functions and regulations. Although cross-linking coupled with mass spectrometry (MS) has been presented as a feasible strategy for structural elucidation of large multisubunit protein complexes, this method has proven challenging because of technical difficulties in unambiguous identification of cross-linked peptides and determination of cross-linked sites by MS analysis. In this work, we developed a novel cross-linking strategy using a newly designed MS-cleavable cross-linker, disuccinimidyl sulfoxide (DSSO). DSSO contains two symmetric collision-induced dissociation (CID)-cleavable sites that allow effective identification of DSSO-cross-linked peptides based on their distinct fragmentation patterns unique to cross-linking types (i.e. interlink, intralink, and dead end). The CID-induced separation of interlinked peptides in MS/MS permits MS(3) analysis of single peptide chain fragment ions with defined modifications (due to DSSO remnants) for easy interpretation and unambiguous identification using existing database searching tools. Integration of data analyses from three generated data sets (MS, MS/MS, and MS(3)) allows high confidence identification of DSSO cross-linked peptides. The efficacy of the newly developed DSSO-based cross-linking strategy was demonstrated using model peptides and proteins. In addition, this method was successfully used for structural characterization of the yeast 20 S proteasome complex. In total, 13 non-redundant interlinked peptides of the 20 S proteasome were identified, representing the first application of an MS-cleavable cross-linker for the characterization of a multisubunit protein complex. Given its effectiveness and simplicity, this cross-linking strategy can find a broad range of applications in elucidating the structural topology of proteins and protein complexes.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Peptídeos
/
Espectrometria de Massas
/
Proteínas
/
Reagentes de Ligações Cruzadas
Tipo de estudo:
Diagnostic_studies
Idioma:
En
Revista:
Mol Cell Proteomics
Assunto da revista:
BIOLOGIA MOLECULAR
/
BIOQUIMICA
Ano de publicação:
2011
Tipo de documento:
Article
País de afiliação:
Estados Unidos