Identification of peptide-binding sites within BSA using rapid, laser-induced covalent cross-linking combined with high-performance mass spectrometry.
J Mol Recognit
; 31(2)2018 02.
Article
en En
| MEDLINE
| ID: mdl-28994207
ABSTRACT
We are developing a rapid, time-resolved method using laser-activated cross-linking to capture protein-peptide interactions as a means to interrogate the interaction of serum proteins as delivery systems for peptides and other molecules. A model system was established to investigate the interactions between bovine serum albumin (BSA) and 2 peptides, the tridecapeptide budding-yeast mating pheromone (α-factor) and the decapeptide human gonadotropin-releasing hormone (GnRH). Cross-linking of α-factor, using a biotinylated, photoactivatable p-benzoyl-L-phenylalanine (Bpa)-modified analog, was energy-dependent and achieved within seconds of laser irradiation. Protein blotting with an avidin probe was used to detect biotinylated species in the BSA-peptide complex. The cross-linked complex was trypsinized and then interrogated with nano-LC-MS/MS to identify the peptide cross-links. Cross-linking was greatly facilitated by Bpa in the peptide, but some cross-linking occurred at higher laser powers and high concentrations of a non-Bpa-modified α-factor. This was supported by experiments using GnRH, a peptide with sequence homology to α-factor, which was likewise found to be cross-linked to BSA by laser irradiation. Analysis of peptides in the mass spectra showed that the binding site for both α-factor and GnRH was in the BSA pocket defined previously as the site for fatty acid binding. This model system validates the use of laser-activation to facilitate cross-linking of Bpa-containing molecules to proteins. The rapid cross-linking procedure and high performance of MS/MS to identify cross-links provides a method to interrogate protein-peptide interactions in a living cell in a time-resolved manner.
Palabras clave
Texto completo:
1
Bases de datos:
MEDLINE
Asunto principal:
Péptidos
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Feromonas
/
Espectrometría de Masas
/
Albúmina Sérica Bovina
/
Hormonas Peptídicas
Tipo de estudio:
Diagnostic_studies
/
Prognostic_studies
Límite:
Animals
/
Humans
Idioma:
En
Revista:
J Mol Recognit
Asunto de la revista:
BIOLOGIA MOLECULAR
Año:
2018
Tipo del documento:
Article
País de afiliación:
Estados Unidos