Abstract 3871: Simultaneous absolute quantitation of ATP-binding cassette transporters in canine tissues via targeted proteomics

Abstract

Abstract The dog serves as a useful model of spontaneously occurring cancer by virtue of similar biology, therapies and mechansims of chemotherapeutic resistance. The ABC transporters are a superfamily of membrane bound proteins that are widely distributed across mammalian tissues and constitute a substantial component of variability in anticancer drug pharmakokinetics and tumor cell resistance. Thus a quantitative atlas of ATP-binding cassette (ABC) transporters across canine tissues is crucial for improving our ability to extrapolate data from dog to human. We have employed a quantitative proteomic approach based on multiple reaction monitoring in tandem mass spectrometry that relies on detection of novel peptide sequences following trypsin digestion of biological samples and separation of peptides utilizing high-pressure liquid chromatography. The 14 proteins evaluated include P-gp, Bcrp, Bsep, Mrp1-6, Mrp7, Mrp9, and the products of canine Abcg5 and Abcg8. Peptide sequences were identified via an in-silico trypsin digestion of proteins and signature peptides were generated for identification and quantitation using stable isotope-labeled signature peptides as an internal reference. Signature peptides were chosen based on lack of posttranslational modification sites and known polymorphisms. Isolated brain microvessels, liver, kidney, duodenum, jejunum, ileum and lung were collected at necropsy from ten normal dogs and membrane fractions of each tissue were prepared. Additional samples were collected for isolation of mRNA. Membrane protein fractions were digested with trypsin and total protein concentrations determined by BCA assay. Calibration curves for signature peptides were prepared in a bovine serum albumin digestate and ranged from 0.25 fmol to 200 fmol on column. Quantitation of peptides in tissue samples was performed via linear regression analysis of calibration curves with extrapolation using at least two transitions for each peptide. Results were obtained as femtomole of peptide per microgram of total protein in the sample. Differential protein levels across tissues were compared to quantitative real time RT-PCR results for the corresponding genes and differences between tissues with each method were concordant. Our results provide the first quantitative atlas of ABC transporters across normal dog tissues and provide a framework for improved understanding of pharmacokinetic and pharmacodynamic variability among dogs receiving anticancer therapies. In addition, this panel will allow evaluation of the potential role that each of these transporters plays in resistance to chemotherapy in vitro and in vivo in dogs, a relevant preclinical model of naturally occurring cancers. Citation Format: Luke A. Wittenburg, Holly Conger, Dominique Ramirez, Daniel Gustafson. Simultaneous absolute quantitation of ATP-binding cassette transporters in canine tissues via targeted proteomics. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3871.