Liquid chromatography/tandem mass spectrometry based targeted proteomics quantification of P‐glycoprotein in various biological samples

Abstract

P-Glycoprotein (P-gp/ABCB1) is expressed in membrane barriers to exclude pharmacological substrates from cells, and therefore influences the ADME/Tox properties and efficacy of therapeutics. In the present study, a liquid chromatography/tandem mass spectrometry (LC/MS/MS)-mediated targeted proteomics was developed to quantitate P-gp protein. With the aid of in silico predictive tools, a unique 9-mer tryptic peptide of P-gp protein was synthesized (with the stable isotope labeled (SIL) peptide as internal standard) and applied for quantitative LC/MS/MS method development. For LC/MS/MS quantification, the N-glycosylation of the peptide, polymorphism and transmembrane region was intended to be excluded during the peptide selection. The lower limit of quantification was established to be 0.025 nM with the linearity of the standard curve ranging to 20 nM of P-gp signature peptides in the matrix digested surrogate bovine serum albumin. The digestion efficiency, both the accuracy (relative error) and the precision (coefficient of variation) of the method, was verified by using the synthetic quantification peptide and the synthetic surrogate substrate peptide that mimics the sequence of tryptic peptide and associated flanking tryptic cleavage sites at the N- and C-terminals. By applying the method developed, the absolute amounts of human, dog and mouse P-gp (Mdr1a) were quantified in various biological samples. LC/MS/MS-mediated P-gp quantification was achieved as a highly sensitive, selective and reproducible assay and could be directly applicable to many current research needs related to P-gp.

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

“…Two unique signature peptides (not present in any other known protein) were selected for quantification of each transporter (Table 1) (Kamiie et al, 2008) and literature reports (Zhang et al, 2011;Balogh et al, 2012). Briefly, peptides with transmembrane regions, single nucleotide polymorphisms (SNPs), posttranslational modifications, or those susceptible to degradation were not selected.…”

Quantitative Targeted Proteomics for Membrane Transporter Proteins: Method and Application

“…The peptides for quantification are easily detectable in an instrument representing the highest responding peptides for the target protein (17). The selection can be accomplished through the use of either empirical experimental tools in a high-resolution mass spectrometry analysis (18), such as time of flight (TOF) and Orbitrap, or in silico predictive tools (7,19) as discussed below. Protein digestion to release peptides can be achieved by a protease reaction with biological samples.…”

“…The proteotypic peptides generated through the digestion of intact protein by different proteases can be predicted through online predictive tools (7). Although there are lots of proteases available, trypsin is most commonly used because it can produce peptides with appropriate amino acid length and composition being amenable for LC-MS detection.…”

“…In SIL methods such as the absolute quantification (AQUA) method (30), the IS is commonly spiked during or postdigestion and is the most feasible and rapid approach. Indeed, SIL peptides are among the most common IS used to evaluate the expression of drug transporters (5)(6)(7)18,19,(46)(47)(48)(49)(50)(51). A comparison of the SIL peptide with SILAM approach was recently carried out in the LC-MS/ MS method development in our laboratory (37).…”

“…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).…”