Fast and Highly Efficient Affinity Enrichment of Azide-A-DSBSO Cross-Linked Peptides.
J Proteome Res
; 19(5): 2071-2079, 2020 05 01.
Article
en En
| MEDLINE
| ID: mdl-32250121
ABSTRACT
Cross-linking mass spectrometry is an increasingly used, powerful technique to study protein-protein interactions or to provide structural information. Due to substochiometric reaction efficiencies, cross-linked peptides are usually low abundance. This results in challenging data evaluation and the need for an effective enrichment. Here we describe an improved, easy to implement, one-step method to enrich azide-tagged, acid-cleavable disuccinimidyl bis-sulfoxide (DSBSO) cross-linked peptides using dibenzocyclooctyne (DBCO) coupled Sepharose beads. We probed this method using recombinant Cas9 and E. coli ribosome. For Cas9, the number of detectable cross-links was increased from â¼100 before enrichment to 580 cross-links after enrichment. To mimic a cellular lysate, E. coli ribosome was spiked into a tryptic HEK background at a ratio of 12-1100. The number of detectable unique cross-links was maintained high at â¼100. The estimated enrichment efficiency was improved by a factor of 4-5 (based on XL numbers) compared to enrichment via biotin and streptavidin. We were still able to detect cross-links from 0.25 µg cross-linked E. coli ribosomes in a background of 100 µg tryptic HEK peptides, indicating a high enrichment sensitivity. In contrast to conventional enrichment techniques, like SEC, the time needed for preparation and MS measurement is significantly reduced. This robust, fast, and selective enrichment method for azide-tagged linkers will contribute to mapping protein-protein interactions, investigating protein architectures in more depth, and helping to understand complex biological processes.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Banco de datos:
MEDLINE
Asunto principal:
Azidas
/
Escherichia coli
Idioma:
En
Revista:
J Proteome Res
Asunto de la revista:
BIOQUIMICA
Año:
2020
Tipo del documento:
Article
País de afiliación:
Austria