Interindividual Variability in Hepatic Organic Anion-Transporting Polypeptides and P-Glycoprotein (ABCB1) Protein Expression: Quantification by Liquid Chromatography Tandem Mass Spectroscopy and Influence of Genotype, Age, and Sex

Abstract

Interindividual variability in protein expression of organic aniontransporting polypeptides (OATPs) OATP1B1, OATP1B3, OATP2B1, and multidrug resistance-linked P-glycoprotein (P-gp) or ABCB1 was quantified in frozen human livers (n = 64) and cryopreserved human hepatocytes (n = 12) by a validated liquid chromatography tandem mass spectroscopy (LC-MS/MS) method. Membrane isolation, sample workup, and LC-MS/MS analyses were as described before by our laboratory. Briefly, total native membrane proteins, isolated from the liver tissue and cryopreserved hepatocytes, were trypsin digested and quantified by LC-MS/MS using signature peptide(s) unique to each transporter. The mean 6 S.D. (maximum/minimum range in parentheses) protein expression (

Quantitative Targeted Proteomics for Membrane Transporter Proteins: Method and Application

“…For example, as summarized in Table I, the amount of human hepatic transporters detected by different laboratories varies significantly. In particular, OATP1B1 was not detected in 9 of 17 livers (85), which did not agree with the reports obtained between different laboratories (12,65). For the reason just specified, the conclusion of expression "atlas" or expression "profile," such as "BCRP was expressed 1.6-fold more than MDR1" (50) or "BCRP was the most abundant followed by MDR1" (48), is particularly concerning under the typical enzymatic digestions employed in these types of studies, which require a balance between conditions that enhance proteolysis through denaturation of the target protein, and conditions that will not significantly inhibit the proteolytic activity of the digestion enzyme.…”

“…It is now known that BCRP expression in the liver with the variant C421A allele was significantly lower than that in the wild-type livers (13). In contrast, OATP1B1 expression in the livers with SLCO1B1 haplotypes *14/*14 and *14/*1a (A388G and C463A variants) was higher than that in the reference haplotype (*1a/*1a) (12), leading to ∼40% decrease in predicted exposure of rosuvastatin or repaglinide in those individuals harboring these variant alleles. Interesting results were also noted as neither age nor sex was associated with expression of hepatic transporters determined (12,13).…”

“…The ability to predict uptake clearance and determine the contribution of individual transporters to overall hepatic uptake is therefore critical in assessing the potential pharmacokinetic and pharmacodynamic variability associated with drug-drug interactions and pharmacogenetics. Since hepatic uptake transporters co- Recently, quantitative targeted proteomics was applied to determine the interindividual variability of expression level of hepatic uptake and efflux transporters in a large set of human liver samples to understand the influence of genotype, age and sex on such expression (12,13). It is now known that BCRP expression in the liver with the variant C421A allele was significantly lower than that in the wild-type livers (13).…”

“…On the other hand, understanding of the specific protease cleavage sites can facilitate the probability of de novo sequencing for a given protein, to predict the peptides that would result from protease digestion of the protein through an in silico digestion. Under the current practice, the combination of in silico analysis and subsequent lab experiments is widely accepted as an effective approach for developing methods to quantify membrane transporters (12,(20)(21)(22).…”

“…It has rapidly advanced and made tremendous impact on a variety of biological fields. Recently, mass spectrometry (MS)-based targeted proteomics with selected reaction monitoring (SRM) has become a promising tool for relative and absolute quantification of proteins (6)(7)(8)(9)(10)(11)(12)(13). Traditionally, this SRM technique was used in conjunction with liquid chromatography (LC) to analyze small molecules such as drugs and their metabolites (14).…”