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Background: Bactrim, the combination antibiotic trimethoprim-sulfamethoxazole (TMP-SMX), is an efficacious and broadly prescribed drug for the treatment of infections. Unfortunately, Bactrim has a high rate of idiosyncratic adverse drug reactions (IADRs), which has almost exclusively been attributed to SMX due to its well-characterized reactive metabolites and protein binding. More recent data suggests that TMP has been overlooked as a cause of these IADRs as TMP can also be bioactivated to reactive intermediates capable of reacting with thiols and potentially proteins [1]. Further studies in which radiolabeled TMP was incubated in vitro with human liver microsomes provided evidence of protein binding by TMP metabolites similar to SMX [2]. Previous in-house studies using anti-TMP antiserum detected circulating TMP protein adducts in patients taking TMP-SMX. However, methods to detect and characterize protein adducts in vivo need to be established. Here we describe the development of methods to enrich and detect TMP-protein adducts in biological matrices. Methods: To investigate whether we could detect TMP adducts in human plasma, we incubated human plasma in vitro with TMP benzylic alcohol metabolite, Cα-OH-TMP, to generate modified proteins. The samples were analyzed by western blotting and liquid chromatography-tandem mass spectrometry (LC-MS/MS) to detect and identify protein adducts. Methods to enrich TMP adducts from biological samples were also investigated. We immobilized partially purified TMP antiserum onto beads, and used these beads to enrich TMP-modified peptides from tryptic digests of HSA incubated with Cα-OH-TMP. The input, flow-through, and eluate of this enrichment were analyzed by LC-MS/MS. Results: We observed modification of plasma proteins by Cα-OH-TMP by western blotting and by LC-MS/MS. Incubation of human plasma with the Cα-OH-TMP metabolite resulted in covalent modification of HSA on the Cys34 residue. Using beads with immobilized TMP antiserum, we were able to selectively enrich TMP-modified peptides from tryptic digests of TMP-modified HSA. Conclusions: We report that Cα-OH-TMP can react with plasma proteins, and that these modified proteins can be detected by LC-MS/MS and western blotting. We also demonstrated a method to enrich TMP-modified peptides from tryptic digests. Further work is required to determine whether these methods can be applied to patient samples to detect and measure specific TMP-modified proteins in vivo. Quantification of TMP-modified proteins in vivo has the potential to yield mechanistic understanding of Bactrim IADRs and to identify prospective biomarkers of IADR development.

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Methods to Detect and Enrich Trimethoprim Protein Adducts in Biological Matrices