Sulfur valence normalization to expand quantitative peptide selection in protein metrology.

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