Microdroplet‐assisted protein adduct formation and characterization by mass spectrometry

Abstract

AbstractThere have been increasing interests in developing proteomics‐based protein adduct detection for monitoring carcinogen exposure risk. Generating adducted protein standards and characterizing the adduction sites by bottom‐up proteomics, however, require lengthy adduction and enzymatic digestion. Here we demonstrated that microdroplet can greatly accelerate the catechol estrogen adduction on hemoglobin (Hb) as well as one‐pot reaction of adduction and enzyme digestion in millisecond for bottom‐up characterization. The adducted Hb generated by microdroplet reaction was characterized to contain one to two catechol estrogens with the β chain of Hb (Hb‐β) by online in situ intact measurement of mass spectrometry. The adduction sites were further identified to be C112‐Hb‐β or C93‐Hb‐β by microdroplet one‐pot or two‐step adduction and digestion. These results were consistent with the intact measurement and also same as the bulk or endogenous reaction. This method is expected to be applicable to prepare protein standards adducted by other reactive oxidizing species to greatly save time and sample amount.