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Title: Identification of peptide-binding sites within BSA using rapid, laser-induced covalent cross-linking combined with high-performance mass spectrometry

Abstract

In this paper, 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 validatesmore » the use of laser-activation to facilitate cross-linking of Bpa-containing molecules to proteins. Finally, 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.« less

Authors:
 [1];  [2];  [3];  [1];  [4]; ORCiD logo [2]; ORCiD logo [1]
+ Show Author Affiliations
  1. Univ. of Tennessee, Knoxville, TN (United States). Dept. of Microbiology
  2. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Chemical Science Division; Univ. of Tennessee, Knoxville, TN (United States). UT‐ORNL Graduate School of Genome Science and Technology
  3. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Chemical Science Division; Univ. of Tennessee, Knoxville, TN (United States). Dept. of Chemistry
  4. CUNY College of Staten Island, New York, NY (United States). Dept. of Chemistry. Macromolecular Assemblies Inst.; The Graduate Center, CUNY, New York, NY (United States). Programs in Biochemistry and Chemistry
Publication Date:
Research Org.:
Oak Ridge National Laboratory (ORNL), Oak Ridge, TN (United States); Univ. of Tennessee, Knoxville, TN (United States)
Sponsoring Org.:
USDOE; National Inst. of Health (NIH) (United States)
OSTI Identifier:
1474715
Grant/Contract Number:  
AC05-00OR22725; GM112496
Resource Type:
Accepted Manuscript
Journal Name:
JMR. Journal of Molecular Recognition
Additional Journal Information:
Journal Volume: 31; Journal Issue: 2; Journal ID: ISSN 0952-3499
Publisher:
Wiley
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; BSA-peptide cross-linking; laser irradiation; mass spectrometry (MS); peptide hormone; peptide pheromone

Citation Formats

Hauser, Melinda, Qian, Chen, King, Steven T., Kauffman, Sarah, Naider, Fred, Hettich, Robert L., and Becker, Jeffrey M. Identification of peptide-binding sites within BSA using rapid, laser-induced covalent cross-linking combined with high-performance mass spectrometry. United States: N. p., 2017. Web. doi:10.1002/jmr.2680.
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Hauser, Melinda, Qian, Chen, King, Steven T., Kauffman, Sarah, Naider, Fred, Hettich, Robert L., & Becker, Jeffrey M. Identification of peptide-binding sites within BSA using rapid, laser-induced covalent cross-linking combined with high-performance mass spectrometry. United States. https://doi.org/10.1002/jmr.2680
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Hauser, Melinda, Qian, Chen, King, Steven T., Kauffman, Sarah, Naider, Fred, Hettich, Robert L., and Becker, Jeffrey M. Tue . "Identification of peptide-binding sites within BSA using rapid, laser-induced covalent cross-linking combined with high-performance mass spectrometry". United States. https://doi.org/10.1002/jmr.2680. https://www.osti.gov/servlets/purl/1474715.
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@article{osti_1474715,
title = {Identification of peptide-binding sites within BSA using rapid, laser-induced covalent cross-linking combined with high-performance mass spectrometry},
author = {Hauser, Melinda and Qian, Chen and King, Steven T. and Kauffman, Sarah and Naider, Fred and Hettich, Robert L. and Becker, Jeffrey M.},
abstractNote = {In this paper, 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. Finally, 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.},
doi = {10.1002/jmr.2680},
journal = {JMR. Journal of Molecular Recognition},
number = 2,
volume = 31,
place = {United States},
year = {Tue Oct 10 00:00:00 EDT 2017},
month = {Tue Oct 10 00:00:00 EDT 2017}
}
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