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Quantification of serine residue stereoinversion in a short peptide by reversed-phase high-performance liquid chromatography: analysis of mechanisms promoting serine stereoinversion

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  • Novel Analytical Technologies Contributing to Clinical and Pharmaceutical Research Fields
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Abstract

Stereoinversion of Ser residues within proteins, which has been identified in long-lived proteins, influences protein function. To quantify the stereoinversion of Ser residues, we investigated the potential adaptation of our direct peptide analytical method originally established for analyzing the isomerization of asparaginyl/aspartyl residues. Peptide pairs containing l-Ser or d-Ser residues with lengths of four or five residues were synthesized. Separation conditions for these peptide pairs were systematically examined by precisely adjusting the pH of the elution solvent using reverse-phase high-performance liquid chromatography (HPLC). Optimal separation conditions were successfully developed for all peptide pairs, enabling the direct quantification of Ser residue stereoinversion through a single HPLC run. Subsequently, the degree of Ser stereoinversion within the model peptide, Gly-Ser-Gly-Tyr, was determined using the method established in this study. Surprisingly, the stereoinversion of Ser residues occurred only when the absolute configurations of Ser and Tyr residues of the peptide differed from each other, whereas no stereoinversion was observed when their absolute configurations were identical. The experiments using peptides similar to the model peptide reveal that both the N-terminal amino group and the hydroxyl group of the C-terminal Tyr residue are involved in the stereoinversion of the Ser residue. By applying a simple method to quantify the stereoinversion of Ser residues, valuable insights into the mechanisms governing these stereoinversions were obtained.

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Data availability

Data are available from the corresponding author upon reasonable request.

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Funding

This work was partially supported by JSPS KAKENHI (Grant number 19K07031 and 23K06088). The authors acknowledge the support, but JSPS KAKENHI had no role in the design of the study, data collection, analysis, interpretation of results, or writing of the manuscript.

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Authors and Affiliations

  1. School of Pharmaceutical Sciences, Suzuka University of Medical Science, Suzuka, 513-8670, Japan

    Yutaka Sadakane, Mizuki Kobayashi, Mitsuteru Sano, Shota Morimoto & Megumi Hagino

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  1. Yutaka Sadakane
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  2. Mizuki Kobayashi
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  3. Mitsuteru Sano
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  4. Shota Morimoto
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  5. Megumi Hagino
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Correspondence to Yutaka Sadakane.

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Sadakane, Y., Kobayashi, M., Sano, M. et al. Quantification of serine residue stereoinversion in a short peptide by reversed-phase high-performance liquid chromatography: analysis of mechanisms promoting serine stereoinversion. ANAL. SCI. 40, 925–934 (2024). https://doi.org/10.1007/s44211-024-00543-5

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