title: Predicted multiple selected reaction monitoring to screen activated drug-mediated modifications on human serum albumin.
abstract: Metabolic activation of drugs frequently generates electrophilic products that may undergo covalent binding to biological macromolecules, such as proteins and DNA. The resulting covalent adducts are of considerable concern in drug discovery and development. Several strategies for assessing the potential risks of candidate drugs have been reported. Of these, glutathione trapping is the most commonly used method together with mass spectrometry. Furthermore, drug-mediated protein modifications have been studied using serum albumin and CYP enzymes to clarify target amino acids and mechanism-based inhibition, respectively. In this article, we introduce a practical way to screen drug-mediated protein modifications. The method, referred to as "predicted multiple selected reaction monitoring," is based on the selected reaction monitoring (SRM) strategy, but targets all possible chemically modified tryptic peptides. The creation of SRM lists may require patience; however, this strategy could facilitate more sensitive screening compared with the common strategy of data-dependent product ion scanning. Ketoprofen-N-hydroxysuccinimidyl ester (equivalent to glucuronide) and N-acetyl-p-benzoquinone imine (NAPQI) were allowed to react with human serum albumin as a model experiment. Using this strategy, 11 ketoprofen-adduction sites (at Lys(137, 195, 199, 212, 351, 402, 432, 436, 525, 536, and 541)) and 1 NAPQI-adduction site (at Cys(34)) were easily identified.
authors.affiliation: Department of Bio-analytical Chemistry, Graduate School of Pharmaceutical Sciences, Tohoku University, Miyagi 980-8578, Japan; Analysis and Pharmacokinetics Research Labs, Astellas Pharma, Inc., Ibaraki 305-8585, Japan.
authors.forename: Fumio
authors.initials: F
authors.lastname: Osaki
authors.affiliation: Department of Bio-analytical Chemistry, Graduate School of Pharmaceutical Sciences, Tohoku University, Miyagi 980-8578, Japan.
authors.forename: Takaaki
authors.initials: T
authors.lastname: Goto
authors.affiliation: Department of Bio-analytical Chemistry, Graduate School of Pharmaceutical Sciences, Tohoku University, Miyagi 980-8578, Japan.
authors.forename: Seon Hwa
authors.initials: SH
authors.lastname: Lee
authors.affiliation: Department of Bio-analytical Chemistry, Graduate School of Pharmaceutical Sciences, Tohoku University, Miyagi 980-8578, Japan. Electronic address: t-oe@mail.pharm.tohoku.ac.jp.
authors.forename: Tomoyuki
authors.initials: T
authors.lastname: Oe
mesh_terms: D000595:Amino Acid Sequence; D016227:Benzoquinones / Q000378:metabolism*; D001665:Binding Sites; D003545:Cysteine / Q000032:analysis / Q000378:metabolism; D006801:Humans; D007097:Imines / Q000378:metabolism*; D007660:Ketoprofen / Q000031:analogs & derivatives* / Q000378:metabolism; D008239:Lysine / Q000032:analysis / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods; D008969:Molecular Sequence Data; D004364:Pharmaceutical Preparations / Q000378:metabolism; D011485:Protein Binding; D012709:Serum Albumin / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D016227:Benzoquinones; D007097:Imines; D004364:Pharmaceutical Preparations; D012709:Serum Albumin; C044223:ketoprofen glucuronide; D007660:Ketoprofen; C028473:N-acetyl-4-benzoquinoneimine; D008239:Lysine; D003545:Cysteine
keywords: Activated drug; Chemical modification; Human serum albumin; Mass spectrometry; Predicted multiple selected reaction monitoring; Screening