Title: | "Identification of peptide-binding sites within BSA using rapid, laser-induced covalent cross-linking combined with high-performance mass spectrometry" |
Author(s): | Hauser M; Qian C; King ST; Kauffman S; Naider F; Hettich RL; Becker JM; |
Address: | "Department of Microbiology, University of Tennessee, Knoxville, TN, USA. Chemical Science Division, Oak Ridge National Laboratory, Oak Ridge, TN, USA. UT-ORNL Graduate School of Genome Science and Technology, University of Tennessee, Knoxville, TN, USA. Department of Chemistry, University of Tennessee, Knoxville, TN, USA. Department of Chemistry and Macromolecular Assemblies Institute, College of Staten Island, CUNY, New York, NY, USA. Programs in Biochemistry and Chemistry, Graduate Center, The City University of New York, New York, NY, USA" |
ISSN/ISBN: | 1099-1352 (Electronic) 0952-3499 (Print) 0952-3499 (Linking) |
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 (alpha-factor) and the decapeptide human gonadotropin-releasing hormone (GnRH). Cross-linking of alpha-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 alpha-factor. This was supported by experiments using GnRH, a peptide with sequence homology to alpha-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 alpha-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" |
Keywords: | "Animals Binding Sites Cattle Humans Mass Spectrometry/*methods Peptide Hormones/*chemistry Peptides/*chemistry Pheromones/*chemistry Protein Binding Serum Albumin, Bovine/*chemistry Tandem Mass Spectrometry BSA-peptide cross-linking laser irradiation mass;" |
Notes: | "MedlineHauser, Melinda Qian, Chen King, Steven T Kauffman, Sarah Naider, Fred Hettich, Robert L Becker, Jeffrey M eng R01 GM112496/GM/NIGMS NIH HHS/ Research Support, N.I.H., Extramural England 2017/10/11 J Mol Recognit. 2018 Feb; 31(2):10.1002/jmr.2680. doi: 10.1002/jmr.2680. Epub 2017 Oct 10" |