Highly sensitive LC-MRM workflow for quantitation of efflux transporters in rat peripheral blood mononuclear cells: leveraging ProteoExcelTP with MRM prediction capability

Although combinational antiretroviral therapy has been proven highly effective, it suffers from drug–drug interactions, drug resistance and adverse reactions with long-term use. The introduction of novel drugs in antiretroviral therapy proposes newer treatment options. However, drug safety and their potential interactions after long-term therapy remain largely unexplored. In this study, the induction potential of bictegravir on efflux transporters at the protein level was assessed by quantifying the transporters using an LC-MS/MS-based method. A surrogate peptide approach was used for the simultaneous determination of P-gp, BCRP and MRP1 transporter proteins in rat peripheral blood mononuclear cells. The previously developed Excel-based ProteoExcel^TP tool was utilized for selecting surrogate peptides corresponding to the target transporters. Moreover, ProteoExcel^TP was integrated with a novel MRM prediction capability for predicting MRM transitions of selected surrogate peptides. The surrogate peptides LLSGQALK (415.2 → 716.4), SSLLDVLAAR (522.8 → 288.1) and EDLDLVLK (472.7 → 685.3) were selected for P-gp, BCRP and MRP1 transporter proteins, respectively. The peptides LLSGQALK, SSLLDVLAAR and EDLDLVLK were eluted at 5.4, 7.0 and 4.1 min, respectively. The findings of the study revealed that bictegravir could significantly induce BCRP transporter after one week of its administration to Sprague-Dawley rats. This finding can be utilized in the future to prevent transporter-mediated drug–drug interactions involving bictegravir. Moreover, the addition of MRM prediction feature to ProteoExcel^TP enhanced its applicability in mass spectrometry-based targeted proteomics. The developed LC-MS/MS-based quantitation method for determining clinically relevant efflux transporters will be useful in investigating the induction potential of other drugs.