Ayano Mori1, Takeshi Masuda1,2, Shingo Ito1,2, Sumio Ohtsuki3,4. 1. Department of Pharmaceutical Microbiology, School of Pharmacy, Kumamoto University, 5-1 Oe-honmachi, Chuo-ku, Kumamoto, 862-0973, Japan. 2. Department of Pharmaceutical Microbiology, Faculty of Life Sciences, Kumamoto University, 5-1 Oe-honmachi, Chuo-ku, Kumamoto, 862-0973, Japan. 3. Department of Pharmaceutical Microbiology, School of Pharmacy, Kumamoto University, 5-1 Oe-honmachi, Chuo-ku, Kumamoto, 862-0973, Japan. sohtsuki@kumamoto-u.ac.jp. 4. Department of Pharmaceutical Microbiology, Faculty of Life Sciences, Kumamoto University, 5-1 Oe-honmachi, Chuo-ku, Kumamoto, 862-0973, Japan. sohtsuki@kumamoto-u.ac.jp.
Abstract
PURPOSE: Quantitative targeted absolute proteomics (QTAP) quantifies proteins by measuring the signature peptides produced from target proteins by trypsin digestion. The selection of signature peptides is critical for reliable peptide quantification. The purpose of this study was to comprehensively assess the digestion efficiency and stability of tryptic peptides and to identify optimal signature peptides for human hepatic transporters and membrane marker proteins. METHODS: The plasma membrane fraction of the human liver was digested at different time points and the peptides were comprehensively quantified using quantitative proteomics. Transporters and membrane markers were quantified using the signature peptides by QTAP. RESULTS: Tryptic peptides were classified into clusters with low digestion efficiency, low stability, and high digestion efficiency and stability. Using the cluster information, we found that a proline residue next to the digestion site or the peptide position in or close to the transmembrane domains lowers digestion efficiency. A peptide containing cysteine at the N-terminus or arginine-glycine lowers peptide stability. Based on this information and the time course of peptide quantification, optimal signature peptides were identified for human hepatic transporters and membrane markers. The quantification of transporters with multiple signature peptides yielded consistent absolute values with less than 30% of coefficient variants in human liver microsomes and homogenates. CONCLUSIONS: The signature peptides selected in the present study enabled the reliable quantification of human hepatic transporters. The QTAP protocol using these optimal signature peptides provides quantitative data on hepatic transporters usable for integrated pharmacokinetic studies.
PURPOSE: Quantitative targeted absolute proteomics (QTAP) quantifies proteins by measuring the signature peptides produced from target proteins by trypsin digestion. The selection of signature peptides is critical for reliable peptide quantification. The purpose of this study was to comprehensively assess the digestion efficiency and stability of tryptic peptides and to identify optimal signature peptides for human hepatic transporters and membrane marker proteins. METHODS: The plasma membrane fraction of the human liver was digested at different time points and the peptides were comprehensively quantified using quantitative proteomics. Transporters and membrane markers were quantified using the signature peptides by QTAP. RESULTS: Tryptic peptides were classified into clusters with low digestion efficiency, low stability, and high digestion efficiency and stability. Using the cluster information, we found that a proline residue next to the digestion site or the peptide position in or close to the transmembrane domains lowers digestion efficiency. A peptide containing cysteine at the N-terminus or arginine-glycine lowers peptide stability. Based on this information and the time course of peptide quantification, optimal signature peptides were identified for human hepatic transporters and membrane markers. The quantification of transporters with multiple signature peptides yielded consistent absolute values with less than 30% of coefficient variants in human liver microsomes and homogenates. CONCLUSIONS: The signature peptides selected in the present study enabled the reliable quantification of human hepatic transporters. The QTAP protocol using these optimal signature peptides provides quantitative data on hepatic transporters usable for integrated pharmacokinetic studies.
Authors: Yi-an Bi; Xi Qiu; Charles J Rotter; Emi Kimoto; Mary Piotrowski; Manthena V Varma; Ayman F Ei-Kattan; Yurong Lai Journal: Biopharm Drug Dispos Date: 2013-10-03 Impact factor: 1.627
Authors: Sieto Bosgra; Evita van de Steeg; Maria L Vlaming; Kitty C Verhoeckx; Maarten T Huisman; Miriam Verwei; Heleen M Wortelboer Journal: Eur J Pharm Sci Date: 2014-09-28 Impact factor: 4.384