An Integrated Strategy\nto Identify Tyrosine Sulfation\nfrom the Therapeutic Proteins

Posttranslational modifications (PTMs) are potential\ncritical quality\nattributes in biotherapeutic development, as they can affect drug\nefficacy and safety. Tyrosine sulfation plays a critical role in protein–protein\ninteractions and has been found on many surface receptors as well\nas antibody complementarity-determining regions (CDR). However, the\npresence and function of tyrosine sulfation in therapeutic proteins\nhave not been broadly investigated due to difficulties in detecting\nthe modification. Here, we establish an integrated strategy to identify\ntyrosine sulfation in biotherapeutic proteins. In silico prediction\nwas used to estimate possible modification sites, followed by the\nelucidation with intact LCMS and native SCX–MS. The combination\nof these three steps takes less than 1 h, which provides quick and\nconfident preliminary detection of potential CQAs. Taking NB1 as an\nexample, three +80 Da mass shifts were observed from intact mass analysis\nand three acidic peaks were monitored by SCX, allowing confirmation\nof modification as either phosphorylation or sulfation. Peptide mapping,\nFe<sup>3+</sup>-IMAC enrichment, and dephosphorylation were further\nconducted to provide improved signal intensity and differentiation\nof modification such as sulfation or phosphorylation. With this integrated\nstrategy, we were able to identify for the first time both tyrosine\nsulfation and serine phosphorylation in one therapeutic protein.