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