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Human Hepatic Transporter Signature Peptides for Quantitative Targeted Absolute Proteomics: Selection, Digestion Efficiency, and Peptide Stability

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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.

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Data Availability

Raw data files of proteomic analysis have been deposited in jPOST (http://jpostdb.org.jPOST ID: JPST001658/PXD034402).

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Funding

This work was supported in part by JSPS KAKENHI (21H02649), JST CREST (JP171024167), and AMED BINDS (22ama121018).

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Authors and Affiliations

  1. Department of Pharmaceutical Microbiology, School of Pharmacy, Kumamoto University, 5-1 Oe-honmachi, Chuo-ku, Kumamoto, 862-0973, Japan

    Ayano Mori, Takeshi Masuda, Shingo Ito & Sumio Ohtsuki

  2. Department of Pharmaceutical Microbiology, Faculty of Life Sciences, Kumamoto University, 5-1 Oe-honmachi, Chuo-ku, Kumamoto, 862-0973, Japan

    Takeshi Masuda, Shingo Ito & Sumio Ohtsuki

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  1. Ayano Mori
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  2. Takeshi Masuda
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  3. Shingo Ito
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  4. Sumio Ohtsuki
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Contributions

All authors contributed to the study design. Mori S conducted the experiments and performed data analysis. Mori S and Ohtsuki S wrote the manuscript. All authors provided final approval for the submitted manuscript.

Corresponding author

Correspondence to Sumio Ohtsuki.

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Conflict of Interest

Sumio Ohtsuki is a full professor at Kumamoto University and is also a director of Proteomedix Frontiers. The other authors declare that they have no conflict of interest.

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Mori, A., Masuda, T., Ito, S. et al. Human Hepatic Transporter Signature Peptides for Quantitative Targeted Absolute Proteomics: Selection, Digestion Efficiency, and Peptide Stability. Pharm Res 39, 2965–2978 (2022). https://doi.org/10.1007/s11095-022-03387-8

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  • DOI: https://doi.org/10.1007/s11095-022-03387-8

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