Synthesis of S-Carbamidomethyl Cysteine and Its Use for Quantification of Cysteinyl Peptides by Targeted Proteomics

Proteomics has played a central role in the identification of reliable disease biomarkers, which are the basis of precision medicine, a promising approach for tackling recalcitrant diseases such as cancer, that elude conventional treatments. Among proteomic methodologies, targeted proteomics employing stable isotope-labeled (SIL) internal standards is particularly suited for the clinical translation of biomarker information owing to its high throughput and accuracy in the quantitative analysis of patient-derived proteomes. Using SIL internal standards ensures the utmost level of confidence in detection and precision in targeted MS experiments. For successfully establishing assays based on targeted proteomics, it is crucial to secure broad coverage when selecting the SIL standard peptide panel. However, cysteinyl peptides have often been excluded because of cysteine's high chemical reactivity. To address this limitation, a new cysteine building block was developed by incorporating a sulfhydryl group configured with an S-carbamidomethyl group, which is commonly used in proteome sampling. This compound was found to be chemically stable and applicable to a variety of solid-phase peptide synthesis (SPPS) campaigns. Furthermore, a direct comparison of the synthesized SIL peptides and tryptic endogenous peptides demonstrated the potential utility of an SPPS flow based on the new cysteine building block for improving the success of targeted proteomic applications.