title: Targeted Mass Spectrometry-Based Approach for Protein-Ligand Binding Analyses in Complex Biological Mixtures Using a Phenacyl Bromide Modification Strategy.
abstract: The characterization of protein folding stability changes on the proteomic scale is useful for protein-target discovery and for the characterization of biological states. The Stability of Proteins from Rates of Oxidation (SPROX) technique is one of several mass spectrometry-based techniques recently established for the making proteome-wide measurements of protein folding and stability. A critical part of proteome-wide applications of SPROX is the identification and quantitation of methionine-containing peptides. Demonstrated here is a targeted mass spectrometry-based proteomics strategy for the detection and quantitation of methionine-containing peptides in SPROX experiments. The strategy involves the use of phenacyl bromide (PAB) for the targeted detection and quantitation of methionine-containing peptides in SPROX using selective reaction monitoring (SRM) on a triple quadrupole mass spectrometer (QQQ-MS). As proof-of-principle, the known binding interaction of Cyclosporine A with cyclophilin A protein in a yeast cell lysate is successfully detected and quantified using a targeted SRM workflow. Advantages of the described workflow over other SPROX protocols include a 20-fold reduction in the amount of total protein needed for analysis and the ability to work with the endogenous proteins in a given sample (e.g., stabile isotope labeling with amino acids in cell culture is not necessary).
authors.affiliation: Department of Chemistry, Duke University , Durham, North Carolina 27708, United States.
authors.forename: Lorrain
authors.initials: L
authors.lastname: Jin
authors.affiliation: Department of Chemistry, Duke University , Durham, North Carolina 27708, United States.
authors.forename: Dongyu
authors.initials: D
authors.lastname: Wang
authors.affiliation: Department of Chemistry, Duke University , Durham, North Carolina 27708, United States.
authors.forename: David M
authors.initials: DM
authors.lastname: Gooden
authors.affiliation: Agilent Technologies, Cary, North Carolina 27511, United States.
authors.forename: Carol H
authors.initials: CH
authors.lastname: Ball
authors.affiliation: Department of Chemistry, Duke University , Durham, North Carolina 27708, United States.
authors.forename: Michael C
authors.initials: MC
authors.lastname: Fitzgerald
mesh_terms: D000098:Acetophenones / Q000737:chemistry*; D001665:Binding Sites; D008024:Ligands; D013058:Mass Spectrometry; D010084:Oxidation-Reduction; D010455:Peptides / Q000737:chemistry; D017510:Protein Folding; D011506:Proteins / Q000737:chemistry*
chemical_list: D000098:Acetophenones; D008024:Ligands; D010455:Peptides; D011506:Proteins; C013190:phenacyl bromide