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J Biomol Screen. 2016 Feb;21(2):136-44. doi: 10.1177/1087057115621288. Epub 2015 Dec 16.

High-Throughput Mass Spectrometric Analysis of Covalent Protein-Inhibitor Adducts for the Discovery of Irreversible Inhibitors: A Complete Workflow.

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Amgen, Molecular Structure and Characterization, Thousand Oaks, CA, USA
Amgen, Discovery Technologies, Thousand Oaks, CA, USA.
Amgen, Information Systems, Cambridge, Thousand Oaks, CA, USA.
Amgen, Medicinal Chemistry, Thousand Oaks, CA, USA.
Amgen, Oncology Research, Thousand Oaks, CA, USA.


We have implemented a solid-phase extraction based time-of-flight mass spectrometer system in combination with novel informatics to rapidly screen and characterize the covalent binding of different irreversible inhibitors to intact proteins. This high-throughput screening platform can be used to accurately detect and quantitate the extent of formation of different covalent protein-inhibitor adducts between electrophilic inhibitors and nucleophilic residues such as cysteine or lysine. For a representative 19.5 kDa protein, the analysis time is approximately 20 s per sample, including an efficient sample loading and desalting step. Accurate protein masses are measured (±0.5 amu of the theoretical molecular weight; measured precision of ±0.02 amu). The fraction of protein reacted with an electrophilic compound is determined relative to an unmodified protein control. A key element of the workflow is the automated identification and quantitation of the expected masses of covalent protein-inhibitor adducts using a custom routine that obviates the need to manually inspect each individual spectrum. Parallel screens were performed on a library of approximately 1000 acrylamide containing compounds (different structures and reactivities) using the solid-phase extraction mass spectrometry based assay and a fluorescence based thiol-reactive probe assay enabling comparison of false positives and false negatives between these orthogonal screening approaches.


deconvolution; electrophile; high-throughput; irreversible inhibitor; multiply charged ions; solid-phase extraction; thiol-probe fluorescence; time-of-flight mass spectrometry

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