Enzyme-Catalyzed Sulfo-Tagging Accelerates 3-OH Steroid-Targeted Submetabolome Profiling

Although derivatization is widely favored for metabolomics, the applications are dramatically narrowed by insufficient selectivity, because a given metabolite may be transferred to several products or conjugated with two or even more derivative moieties. Enzymatic derivatization may address this critical issue by attributing to superior selectivity. Human sulfotransferase 2A1 (hSULT 2A1) was utilized here to tag sulfo to 3-OH of steroids that structurally involve most cholesterol metabolites and, importantly, serve as key biomarkers for diverse diseases. Through evaluating sulfation performances by assaying 53 authentic steroids, we found: 1) great selectivity and transformation rate (>80%) existed for 3-OH sulfation; 2) sulfates exhibited diagnostic fragment ions (i.e., SO₃^-• and SO₄^-), SO₃ neutral loss, and [³⁴S - M - H]^- signals; 3) optimal collision energy for either SO₃^-• or SO₄^- was linearly correlated with [M - H]^- mass; and 4) better sensitivity appeared for sulfates. Molecular docking consolidated selective 3-OH sulfation. hSULT 2A1-catalyzed sulfo-tagging was applied for 3-OH steroid-targeted submetabolome profiling of <i>Bufonis Venenum</i> (BV), a promising anticancer agent. Sixty steroid 3-sulfates were captured and quantitatively compared, and significant variations existed within 20 batches of BV. Together, hSULT 2A1-mediated sulfation is meaningful for submetabolomics targeting on 3-OH steroids, leading to new insights toward enzyme-catalyzed derivative metabolomics.