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Liquid chromatography/tandem mass spectrometry based targeted proteomics quantification of P‐glycoprotein in various biological samples
Author(s) -
Zhang Yiqun,
Li Na,
Brown Paul W.,
Ozer Josef S.,
Lai Yurong
Publication year - 2011
Publication title -
rapid communications in mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.528
H-Index - 136
eISSN - 1097-0231
pISSN - 0951-4198
DOI - 10.1002/rcm.5026
Subject(s) - chemistry , peptide , chromatography , tandem mass spectrometry , quantitative proteomics , mass spectrometry , liquid chromatography–mass spectrometry , tandem mass tag , proteomics , label free quantification , selected reaction monitoring , biochemistry , gene
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. Copyright © 2011 John Wiley & Sons, Ltd.