Characterizing protein modifications by reactive metabolites using magnetic bead bioreactors and LC-MS/MS

Li, Dandan; Fu, You‐Jun; Rusling, James F.

doi:10.1039/c5cc00420a

Cited by 3 publications

(7 citation statements)

References 24 publications

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“…These results confirm NAPQI formation in our system. About 10% of acetaminophen converts to NAPQI, but major metabolites are nontoxic glucuronide (acetaminophen-gluc) and sulfate (acetaminophen-sulf) as found in humans. , …”

Section: Resultsmentioning

confidence: 99%

“…Layer-by-layer (LbL) assembly of films of microsomes and cytochrome p450 supersomes on magnetic beads (MB/PDDA/PSS/PDDA/enzymes) were similar to those described previously. − Full details are described in the SI file.…”

Section: Experimental Sectionmentioning

confidence: 99%

“…We previously identified and detected hGSTP–NAPQI adducts by using magnetic beads (MBs) coated with cyt P450 2E1 as bioreactors . In the present paper, we extend this approach to multiple-organ microsomal and supersomal enzyme sources to evaluate relative amounts of NAPQI–GSH conjugate.…”

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confidence: 97%

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Organ-Specific Screening for Protein Damage Using Magnetic Bead Bioreactors and LC–MS/MS

et al. 2020

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A new 96-well plate methodology for fast, enzyme-multiplexed screening for metabolite−protein adducts was developed. Magnetic beads coated with metabolic enzymes were used to make potentially reactive metabolites that can react with test protein in the wells, followed by sample workup in multiple 96-well filter plates for LC−MS/MS analysis. Incorporation of human microsomes from multiple organs and selected supersomes of single cytochrome P450 (cyt P450) enzymes on the magnetic beads provided a broad spectrum of metabolic enzymes. The reacted protein was then isolated, denatured, reduced, alkylated, and digested, and peptides were collected in a sequence of 96-well filter plates for analysis. Method performance was evaluated by trapping acetaminophen reactive metabolite N-acetylp-benzoquinoneimine (NAPQI) with human glutathione S-transferase pi (hGSTP), human serum albumin (HSA), and bovine serum albumin (BSA) as model target proteins. Relative amounts of acetaminophen metabolite and hGSTP adducts were compared with 10 different cyt P450 enzymes. Human liver microsomes and CYP1A2 supersomes showed the highest bioactivation rate for adduct formation, in which all four cysteines of hGSTP reacted with NAPQI. Eight cysteines of HSA and four cysteines of BSA have been detected to react with NAPQI. This method has the potential for fast multienzyme protein adduct screening with high efficiency and accuracy.

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Section: Resultsmentioning

confidence: 99%

Section: Experimental Sectionmentioning

confidence: 99%

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Organ-Specific Screening for Protein Damage Using Magnetic Bead Bioreactors and LC–MS/MS

et al. 2020

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“…[Color figure can be viewed at wileyonlinelibrary.com] often correlate with adverse effects, such as tissue injury and allergy. 10,11 However, some metabolites showed active potential, and some even had better activity than prototypes. 12 The dual role of metabolite−protein adducts reminds us that we cannot neglect the identification of metabolite modifications.…”

Section: Introductionmentioning

confidence: 99%

“…Moreover, metabolites can modify proteins and induce biological effects. The unexpected modifications caused by metabolites often correlate with adverse effects, such as tissue injury and allergy 10,11 . However, some metabolites showed active potential, and some even had better activity than prototypes 12 .…”

Section: Introductionmentioning

confidence: 99%

Dual roles of drug or its metabolite−protein conjugate: Cutting‐edge strategy of drug discovery using shotgun proteomics

Gong

Chen

Sun

et al. 2022

Medicinal Research Reviews

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Many drugs can bind directly to proteins or be bioactivated by metabolizing enzymes to form reactive metabolites (RMs) that rapidly bind to proteins to form drug−protein conjugates or metabolite−protein conjugates (DMPCs). The close relationship between DMPCs and idiosyncratic adverse drug reactions (IADRs) has been recognized; drug discovery teams tend to avoid covalent interactions in drug discovery projects. Covalent interactions in DMPCs can provide high potency and long action duration and conquer the intractable targets, inspiring drug design, and development. This forms the dual role feature of DMPCs. Understanding the functional implications of DMPCs in IADR control and therapeutic applications requires precise identification of these conjugates from complex biological samples. While classical biochemical methods have contributed significantly to DMPC detection in the past decades, the low abundance and low coverage of DMPCs have become a bottleneck in this field. An emerging transformation toward shotgun proteomics is on the rise. The evolving shotgun proteomics techniques offer improved reproducibility, throughput, specificity, operability, and standardization. Here, we review recent progress in the systematic discovery of DMPCs using shotgun proteomics. Furthermore, the applications of shotgun proteomics supporting drug development, toxicity mechanism investigation, and drug repurposing processes are also reviewed and prospected.

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On-chip signal amplification of magnetic bead-based immunoassay by aviating magnetic bead chains

Jalal

Jin

Eom

et al. 2018

Bioelectrochemistry

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Characterizing protein modifications by reactive metabolites using magnetic bead bioreactors and LC-MS/MS

Abstract: We report here label-free metabolite-protein adduct detection and identification employing magnetic beads coated with metabolic enzymes as bioreactors to generate metabolites and possible metabolite-protein adducts for analysis by liquid chromatography-tandem mass spectrometry.

Cited by 3 publications

References 24 publications

Organ-Specific Screening for Protein Damage Using Magnetic Bead Bioreactors and LC–MS/MS

Organ-Specific Screening for Protein Damage Using Magnetic Bead Bioreactors and LC–MS/MS

Dual roles of drug or its metabolite−protein conjugate: Cutting‐edge strategy of drug discovery using shotgun proteomics

On-chip signal amplification of magnetic bead-based immunoassay by aviating magnetic bead chains

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