Targeted Mass Spectrometry-Based Approach for Protein-Ligand Binding Analyses in Complex Biological Mixtures Using a Phenacyl Bromide Modification Strategy.


Journal Article

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).

Full Text

Duke Authors

Cited Authors

  • Jin, L; Wang, D; Gooden, DM; Ball, CH; Fitzgerald, MC

Published Date

  • November 2016

Published In

Volume / Issue

  • 88 / 22

Start / End Page

  • 10987 - 10993

PubMed ID

  • 27740755

Pubmed Central ID

  • 27740755

Electronic International Standard Serial Number (EISSN)

  • 1520-6882

International Standard Serial Number (ISSN)

  • 0003-2700

Digital Object Identifier (DOI)

  • 10.1021/acs.analchem.6b02658


  • eng