title: High-Throughput Mass Spectrometric Analysis of Covalent Protein-Inhibitor Adducts for the Discovery of Irreversible Inhibitors: A Complete Workflow.
abstract: 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.
authors.affiliation: Amgen, Molecular Structure and Characterization, Thousand Oaks, CA, USA iainc@amgen.com.
authors.forename: Iain D G
authors.initials: ID
authors.lastname: Campuzano
authors.affiliation: Amgen, Discovery Technologies, Thousand Oaks, CA, USA.
authors.forename: Tisha
authors.initials: T
authors.lastname: San Miguel
authors.affiliation: Amgen, Information Systems, Cambridge, Thousand Oaks, CA, USA.
authors.forename: Todd
authors.initials: T
authors.lastname: Rowe
authors.affiliation: Amgen, Information Systems, Cambridge, Thousand Oaks, CA, USA.
authors.forename: Daniel
authors.initials: D
authors.lastname: Onea
authors.affiliation: Amgen, Medicinal Chemistry, Thousand Oaks, CA, USA.
authors.forename: Victor J
authors.initials: VJ
authors.lastname: Cee
authors.affiliation: Amgen, Oncology Research, Thousand Oaks, CA, USA.
authors.forename: Tara
authors.initials: T
authors.lastname: Arvedson
authors.affiliation: Amgen, Discovery Technologies, Thousand Oaks, CA, USA.
authors.forename: John D
authors.initials: JD
authors.lastname: McCarter
mesh_terms: D020106:Acrylamide / Q000737:chemistry*; D003545:Cysteine / Q000737:chemistry; D057166:High-Throughput Screening Assays / Q000379:methods; D008239:Lysine / Q000737:chemistry; D013058:Mass Spectrometry / Q000379:methods; D011506:Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry*; D052616:Solid Phase Extraction / Q000379:methods
chemical_list: D011506:Proteins; D020106:Acrylamide; D008239:Lysine; D003545:Cysteine
keywords: deconvolution; electrophile; high-throughput; irreversible inhibitor; multiply charged ions; solid-phase extraction; thiol-probe fluorescence; time-of-flight mass spectrometry