Targeted Mass Spectrometry-Based Approach for Protein-Ligand Binding Analyses in Complex Biological Mixtures Using a Phenacyl Bromide Modification Strategy
Targeted Mass Spectrometry-Based Approach for Protein-Ligand Binding Analyses in Complex Biological Mixtures Using a Phenacyl Bromide Modification Strategy
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DOI：
10.1021/acs.analchem.6b02658
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发表时间：
2016-11-15
影响因子：
7.4
通讯作者：
Fitzgerald, Michael C.
中科院分区：
文献类型：
--
作者：
Jin, Lorrain;Wang, Dongyu;Fitzgerald, Michael C.
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).
