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

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.

Bifunctional Nano‐Assembly of Iridium(III) Phthalocyanine Complex Encapsulated with BSA: Hypoxia‐relieving/Sonosensitizing Effects and their Immunogenic Sonodynamic Therapy

“…To improve its water solubility and biocompatibility, IrPc complex was encapsulated with BSA to form IrPc nanoparticles (IrPc-NPs) by using the reported protocol that is a good carrier for cancer drugs to prepare nanoparticles (IrPc-NPs) according to the previous reports. [19] The content of IrPc complex in the IrPc-NPs was estimated as 2.25% (w/w) by using UV-vis spectroscopy(Figure S3, Supporting Information). The absorption spectrum of IrPc-NPs in an aqueous solution exhibited a broad and relatively low absorption in the Q band, which was located at ≈705 nm (Figure 1c), while a sharp and intense absorption in DMF at ∼698 nm was observed (Figure S3, Supporting Information), suggesting IrPc was aggregated coated with BSA in water.…”

UV Laser-Induced, Time-Resolved Transcriptome Responses ofSaccharomyces cerevisiae

“…Under these conditions DNA damage was expected to be minimal in contrast to radiation at 254nm that is commonly used for radiation-induced DNA damage in yeast (Guintini et al 2017; Li et al 2018). The strong laser output we used allowed for a much shorter exposure time (30 sec) and a longer wavelength (355nm) that corresponded to the conditions used for our previous protein-peptide cross-linking experiments (Hauser et al 2018).…”

“…In a previous study using UV-laser irradiation, we induced a rapid cross-linking of a peptide labeled with photoactivatable amino acid analog p -benzoyl-L-phenylalanine (Bpa) into a binding pocket of bovine serum albumin (Hauser et al 2018). This protocol allowed cross-linking of the ligand to the substrate in a matter of seconds, in contrast to longer exposure required under a conventional UV lamp.…”

A High-Performance Workflow for Identifying Site-Specific Crosslinks Originating from a Genetically Incorporated, Photoreactive Amino Acid

“…For each of the smaller databases, StavroX identified 10 or fewer crosslink PSMs and MeroX identified 32 crosslink PSMs. The small number of PSMs was surprising given that StavroX [19][20][21][22][23] and MeroX 24,25 have been used to identify site-specific BPA crosslinks in previous studies and https://doi.org/10.26434/chemrxiv-2024-33v24-v2 ORCID: https://orcid.org/0000-0003-3526-4973 Content not peer-reviewed by ChemRxiv. License: CC BY-NC-ND 4.0 because we enriched crosslinks by only excising the band for the crosslinked HSPB5-HSPB5 dimeric product prior to in-gel digestion.…”