Simultaneous Absolute Quantification of 11 Cytochrome P450 Isoforms in Human Liver Microsomes by Liquid Chromatography Tandem Mass Spectrometry with In Silico Target Peptide Selection

https://doi.org/10.1002/jps.22255Get rights and content

ABSTRACT

Cytochrome P450 (CYP) proteins are involved in the biological oxidation and reduction of xenobiotics, affecting the pharmacological efficiency of drugs. This study aimed to establish a method to simultaneously quantify 11 CYP isoforms by multiplexed-multiple reaction monitoring analysis with liquid chromatography tandem mass spectrometry and in silico peptide selection to clarify CYP isoform expression profiles in human liver tissue. CYP1A2, 2A6, and 2D6 target peptides were identified by shot-gun proteomic analysis, and those of other isoforms were selected by in silico peptide selection criteria. The established quantification method detected target peptides at 10 fmol, and the dynamic range of calibration curves was at least 500-fold. The quantification value of CYP1A2 in Supersomes was not significantly different between the established method and quantitative immunoblot analysis. The absolute protein expression levels of 11 CYP isoforms were determined from one pooled and 10 individual human liver microsomes. In the individual microsomes, CYP2C9 showed the highest protein expression level, and CYP1A2, 2A6, 2C19, and 3A4 protein expression exhibited more than a 20-fold difference among individuals. This highly sensitive and selective quantification method is a useful tool for the analysis of highly homologous CYP isoforms and the contribution made by each CYP isoform to drug metabolism.

Section snippets

Abbreviations:

    CYP

    cytochrome P450

    EMS

    enhanced mass scan

    EPI

    enhanced product ion

    ER

    enhanced resolution

    HLM

    human liver microsome

    LC-MS/MS

    liquid chromatography tandem mass spectrometry

    MRM

    multiple reaction monitoring

    MS

    mass spectrometry.

INTRODUCTION

Cytochrome P450 (CYP) is one of the largest families of proteins, consisting of 57 putatively functional genes whose products carry out a wide range of biological oxidations and reductions in the metabolism of drugs, xenobiotics, and endogenous compounds.1 The main CYP proteins expressed in human liver are CYP1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4, and their expression and activities dictate the pharmacological effect of drugs.2 To date, antibodies have been used for CYP protein

Reagents

All peptides (Tab. 2) were synthesized by Thermoelectron Corporation (Sedanstrabe, Germany). Supersomes, which are microsomes derived from insect High-five cells (derived from Trichoplusia ni) overexpressing one of the human isoforms (CYP1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 3A4, or 3A5) were purchased from BD Biosciences (Oxford, UK). Pooled HLMs (HLM610) was purchased from XenoTech, L.L.C. (Kansas City, KS). Other chemicals were commercial products of analytical grade.

Identification of Proteotypic Peptides by Shot-Gun Analysis

Supersomes were suspended in 100

Identification of Target Peptides for Quantifying CYP Isoforms by Shot-Gun Proteomic Analysis

The present study performed shot-gun proteomic analysis to identify target peptides specific for each CYP isoform in trypsin-digested Supersomes, which are microsomes of High-five cells overexpressing one of the following human CYP isoforms: CYP1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 3A4, or 3A5. Table 1 shows the amino acid sequences of identified proteotypic peptides by shot-gun proteomic analysis using nanoLC-MS/MS. Although 2–10 peptides were identified for each isoform examined, specific

DISCUSSION

MRM-based quantification allows a highly sensitive and selective quantification of targeted proteins by measuring target peptides.13,14 The most important issue for this quantification is selection of the target peptide, which must be specific for the target protein and provide sufficient intensity for LC-MS/MS analysis.15 The present study employed two distinct selection methods. The first was the identification of proteotypic peptides from trypsin-digested samples by shot-gun proteomic

ACKNOWLEDGEMENTS

This study was supported in part by the Grant for Development of Creative Technology Seeds Supporting Program for Creating University Ventures from Japan Science and Technology Agency (JST), and the Industrial Technology Research Grant Program from New Energy and the Industrial Technology Development Organization (NEDO) of Japan.

REFERENCES (28)

  • B. Domon et al.

    Mass spectrometry and protein analysis

    Science

    (2006)
  • M.Z. Wang et al.

    A gel-free MS-based quantitative proteomic approach accurately measures cytochrome P450 protein concentrations in human liver microsomes

    Proteomics

    (2008)
  • E. Langenfeld et al.

    Mass spectrometry-based absolute quantification of microsomal cytochrome P450 2D6 in human liver

    Proteomics

    (2009)
  • C. Seibert et al.

    Multiple-approaches to the identification and quantification of cytochromes P450 in human liver tissue by mass spectrometry

    J Proteome Res

    (2009)
  • Cited by (145)

    • Application of reaction phenotyping to address pharmacokinetic variability in patient populations

      2023, Overcoming Obstacles in Drug Discovery and Development: Surmounting the Insurmountable-Case Studies for Critical Thinking
    • Drug Metabolism: Cytochrome P450

      2022, Comprehensive Pharmacology
    View all citing articles on Scopus
    View full text