Quantitative Targeted Proteomics for Membrane Transporter Proteins: Method and Application

Abstract

Abstract. Although global proteomics has shown promise for discovery of many new proteins, biomarkers, protein modifications, and polymorphisms, targeted proteomics is emerging in the proteomics research field as a complement to untargeted shotgun proteomics, particularly when a determined set of low-abundance functional proteins need to be measured. The function and expression of proteins related to drug absorption, distribution, metabolism, and excretion (ADME) such as cytochrome P450 enzymes and membrane transporters are of great interest in biopharmaceutical research. Since the variation in ADME-related protein expression is known to be a major complicating factor encountered during in vitro-in vivo and in vivo-in vivo extrapolations (IVIVE), the accurate quantification of the ADME proteins in complex biological systems becomes a fundamental element in establishing IVIVE for pharmacokinetic predictions. In this review, we provide an overview of relevant methodologies followed by a summary of recent applications encompassing mass spectrometry-based targeted quantifications of membrane transporters.KEY WORDS: drug absorption, distribution, metabolism, and excretion (ADME); drug transporters; in vitro-in vivo and in vivo-in vivo extrapolations (IVIVE); LC-MS/MS; quantitative targeted proteomics.

Development of a Rat Plasma and Brain Extracellular Fluid Pharmacokinetic Model for Bupropion and Hydroxybupropion Based on Microdialysis Sampling, and Application to Predict Human Brain Concentrations

“…However, the low occupancy reported in these studies is below that which our predictions would suggest (approximately 50%) based on the simulated concentrations being similar to the reported IC 50 for hydroxybupropion at this transporter (630 nM; Lukas et al, 2010). This difference identifies the need for studies to investigate the mechanism of bupropion and hydroxybupropion transport across blood-brain surrogate models and, if a transporter is involved, to apply mass spectrometry-based proteomics to support interspecies scaling (Qiu et al, 2014). The predicted exposures for bupropion and hydroxybupropion, together with their IC 50 values for NET, indicate that, if norepinephrine levels are increased in humans via NET inhibition, this would largely be attributed to hydroxybupropion, and not bupropion.…”

“…The model did not include brain ECF bulk flow, since the estimated value of 0.18 to 0.29 ml/min × g brain (Szentistványi et al, 1984) is well below 1% of the CL out estimates and thus is a negligible contributing factor to brain clearance. The utility of modeling plasma and brain distribution across the BBB is that this approach converts an observation (K p,uu ) into parameters (CL in and CL out ) that correlate with physiologic carrier-mediated processes that are potentially scalable between species (Qiu et al, 2014), and whose function could be sensitive to intersubject variability caused by disease or genetic variability as well as drug-drug and drug-metabolite interactions.…”

In Vitro-In Vivo Extrapolation Scaling Factors for Intestinal P-Glycoprotein and Breast Cancer Resistance Protein: Part I: A Cross-Laboratory Comparison of Transporter-Protein Abundances and Relative Expression Factors in Human Intestine and Caco-2 Cells

“…In fact, several laboratories foster links with the forerunning developers of these methodologies, i.e., Tohoku University (Sendai, Japan) and Pfizer Ltd. (Groton, CT). Peptides that are used as surrogates for the quantification of the complete protein are selected on the basis of a variety of criteria Oswald et al, 2013;Prasad and Unadkat, 2014b;Qiu et al, 2014). Consequently, for the frequently quantified transporter-protein P-gp, three peptides are routinely employed to quantify human P-gp (AGAVAEEVLAAIR, NTTGALTTR, FYD-PLAGK; Table 2), highlighting a current lack of consensus among groups.…”

Network based subcellular proteomics in monocyte membrane revealed novel candidate genes involved in osteoporosis

“…However, presenting an exact map of subcellular distribution of all proteins under different conditions is challenging, because protein subcellular localization is dynamic, without strict limitations or boundaries [31]. In particular, membrane or transmembrane proteins are often in extremely low abundance but play multiple and significant roles as signal receptors, enzymes, transport proteins, biomarkers, and cell adhesion molecules [32]. In this study, by using an efficient and reliable subcellular extraction kit in conjunction with high sensitivity and resolution LC-MS instruments, we performed comprehensive proteomics profiling in human monocyte membrane.…”