Analysis of ligand-receptor cross-linked fragments by mass spectrometry.
G-protein coupled receptors (GPCRs) are a class of integral membrane receptor proteins that are characterized by a signature seven-transmembrane (7-TM) configuration. The alpha-factor receptor (Ste2p) from Saccharomyces cerevisiae is a GPCR that, upon binding of a peptide ligand, transduces a signal to initiate a cascade of events leading to the mating of haploid yeast cells. This study summarizes the application of affinity purification and of matrix-assisted laser-desorption ionization time-of-flight (MALDI-TOF) experiments using biotinylated photoactivatable alpha-factor analogs. Affinity purification and enrichment of biotinylated peptides by monomeric avidin beads resulted in mass spectrometric detection of specific signals corresponding to cross-linked fragments of Ste2p. Data obtained from cyanogen bromide (CNBr) fragments of receptor cross-linked to an alpha-factor analog with the photoaffinity group p-benzoyl-l-phenylalanine on position 1 were in agreement with the previous results reported by our laboratory suggesting the cross-linking between position 1 of alpha-factor and a region of Ste2p covering residues 251-294.
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C.D. Son and J.M. Becker, Department of Microbiology, University of Tennessee, Knoxville, TN 37996-0845, USA.
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D001360:Avidin / Q000737:chemistry; D001665:Binding Sites; D019921:Biotinylation; D003432:Cross-Linking Reagents; D008024:Ligands; D013058:Mass Spectrometry; D010446:Peptide Fragments / Q000737:chemistry; D019920:Photoaffinity Labels; D051964:Receptors, Mating Factor; D018000:Receptors, Peptide / Q000737:chemistry* / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D019809:Streptavidin / Q000737:chemistry; D014157:Transcription Factors / Q000737:chemistry* / Q000378:metabolism*
D003432:Cross-Linking Reagents; D008024:Ligands; D010446:Peptide Fragments; D019920:Photoaffinity Labels; D051964:Receptors, Mating Factor; D018000:Receptors, Peptide; D014157:Transcription Factors; D001360:Avidin; D019809:Streptavidin