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<p>Post-translational modifications (PTMs) play a critical role in regulating protein structure and function, and their accurate quantification is essential for disease research and clinical diagnostics. However, the presence of multiple and heterogeneous modifications often interferes with precise quantification of specific PTMs. A major challenge arises from methionine residues, which are highly susceptible to oxidation during sample preparation and analysis, leading to poor quantitative reproducibility with relative standard deviations (RSDs) frequently exceeding 15%. As a result, peptides containing methionine are typically excluded from use as quantitative peptides. This exclusion, however, can severely limit quantitative coverage, particularly when functionally important PTM sites are proximal to methionine residues. To address this limitation, we developed a robust strategy to eliminate the impact of methionine oxidation by normalizing sulfur element valence states. Two characteristic peptides from α-S2-casein, each containing both a phosphorylation site and a methionine residue, were selected as model analytes. Methionine residues were intentionally oxidized using hydrogen peroxide (H<sub>2</sub>O<sub>2</sub>), and key reaction parameters, including oxidation time, temperature, and oxidant concentration, were systematically optimized to ensure complete and consistent oxidation. This controlled oxidation effectively stabilized methionine-containing peptides and eliminated variability arising from uncontrolled oxidation. Using the optimized method, the phosphorylation occupancies of the two α-S2-casein peptides were determined to be 98.72% and 0.42%, respectively. Importantly, the RSDs of the quantitative results were dramatically reduced to 0.24% and 1.42%, demonstrating a substantial improvement in measurement precision. This approach is straightforward, reproducible, and highly effective, enabling accurate quantification of methionine-containing peptides. By mitigating methionine-induced variability, the method significantly broadens the selection range of quantitative peptides and enhances the reliability of protein metrology by mass spectrometry.</p>

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Sulfur valence normalization to expand quantitative peptide selection in protein metrology

  • 期刊论文

  • 发表日期：

    2026年6月26日

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  • Wenhui Fang,
  • Manman Zhu,
  • Wenxin Qiu,
  • Yuan Liu,
  • Kai Sun,
  • Zhanying Chu,
  • Rui Zhai,
  • Xiang Fang

摘要

Post-translational modifications (PTMs) play a critical role in regulating protein structure and function, and their accurate quantification is essential for disease research and clinical diagnostics. However, the presence of multiple and heterogeneous modifications often interferes with precise quantification of specific PTMs. A major challenge arises from methionine residues, which are highly susceptible to oxidation during sample preparation and analysis, leading to poor quantitative reproducibility with relative standard deviations (RSDs) frequently exceeding 15%. As a result, peptides containing methionine are typically excluded from use as quantitative peptides. This exclusion, however, can severely limit quantitative coverage, particularly when functionally important PTM sites are proximal to methionine residues. To address this limitation, we developed a robust strategy to eliminate the impact of methionine oxidation by normalizing sulfur element valence states. Two characteristic peptides from α-S2-casein, each containing both a phosphorylation site and a methionine residue, were selected as model analytes. Methionine residues were intentionally oxidized using hydrogen peroxide (H2O2), and key reaction parameters, including oxidation time, temperature, and oxidant concentration, were systematically optimized to ensure complete and consistent oxidation. This controlled oxidation effectively stabilized methionine-containing peptides and eliminated variability arising from uncontrolled oxidation. Using the optimized method, the phosphorylation occupancies of the two α-S2-casein peptides were determined to be 98.72% and 0.42%, respectively. Importantly, the RSDs of the quantitative results were dramatically reduced to 0.24% and 1.42%, demonstrating a substantial improvement in measurement precision. This approach is straightforward, reproducible, and highly effective, enabling accurate quantification of methionine-containing peptides. By mitigating methionine-induced variability, the method significantly broadens the selection range of quantitative peptides and enhances the reliability of protein metrology by mass spectrometry.