Detection of Protein Polysulfidation Using a β-(4-Hydroxyphenyl)ethyl Iodoacetamide-Derived Biotin Tag HPB

Protein polysulfidation (Pr-S<i>_n</i>H, <i>n</i> ≥ 2) is a type of post-translational modification that plays multiple physiological functions. Detecting Pr-S_<i>n</i>H remains challenging due to the high reactivity of -S<i>_n</i>H groups and their similarity to other thiol-based modifications. Here, we report the development of a new biotinylated tag, HPB, which is derived from β-(4-hydroxyphenyl)ethyl iodoacetamide (HPE-IAM). HPB demonstrates superior performance over the traditional iodoacetamide (IAM)-derived biotin tag IAB, especially in minimizing off-target alkylation and preserving polysulfide chains. After protocol optimization, we achieved a 73.3% accuracy rate in proteomic scale Pr-S<i>_n</i>H detection. Further, we found that polysulfides can modify other amino acids besides cysteine, including histidine, phenylalanine, and tryptophan, by introducing -SH or -SSH groups into their heterocyclic/phenyl rings. This leads to the detection accuracy rate of all current methods being less than 100%. Nonetheless, this study provides a reliable tool for detecting protein polysulfidation from complex cellular backgrounds.