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A tag‐free collisionally induced fragmentation approach to detect drug‐adducted proteins by mass spectrometry
Author(s) -
Ballard T. Eric,
Dahal Upendra P.,
Bessire Andrew J.,
Schneider Richard P.,
Geoghegan Kieran F.,
Vaz Alfin D. N.
Publication year - 2015
Publication title -
rapid communications in mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.528
H-Index - 136
eISSN - 1097-0231
pISSN - 0951-4198
DOI - 10.1002/rcm.7375
Subject(s) - chemistry , small molecule , adduct , mass spectrometry , fragmentation (computing) , molecule , collision induced dissociation , tandem mass spectrometry , combinatorial chemistry , chromatography , biochemistry , organic chemistry , computer science , operating system
Rationale The covalent modification of proteins by toxicants, new chemical entities or drug molecules, either by metabolic activation or the presence of inherently reactive functional groups, is commonly implicated in organ toxicity and idiosyncratic reactions. In efforts to better prosecute protein modifications, we investigated a tag‐free technique capable of detecting protein‐small molecule adducts based solely on the collision‐induced dissociation (CID) of the protein‐small molecule complex. Detection of proteins using unique CID small molecule (SM) product ions would mitigate common issues associated with tagging technologies (e.g., altered reactivity/affinity of the protein‐SM complex). Methods A Waters SYNAPT G2 mass spectrometer (MS) was operated in MS e mode with appropriate collision energy conditions during the MS 2 acquisition for fragmentation of protein‐small molecule adducts to generate characteristic small molecule product ions. Results Ibrutinib, an acrylamide‐containing small molecule drug, was shown to form adducts with rat serum albumin in ex vivo experiments and these adducts were detected by relying solely on the CID product ions generated from ibrutinib. Additionally, ibrutinib produced three CID product ions, one of which was a selective protein‐ibrutinib fragment ion not produced by the compound alone. Conclusions Herein we describe a tag‐free mass spectral detection technique for protein‐small molecule conjugates that relies on the unique product ion fragmentation profile of the small molecule. This technique allows the detection of macromolecular ions containing the adducted small molecule from complex protein matrices through mass range selection for the unique product ions in the CID spectra. Copyright © 2015 John Wiley & Sons, Ltd.