Ma, Cheng-HuaCheng-HuaMaChen, Chih-LinChih-LinChenCHENG-CHIH HSU2023-11-062023-11-062023-10-1913597345https://scholars.lib.ntu.edu.tw/handle/123456789/636828Enzymatic digestion remains one of the "rate-determining steps" in the bottom-up analysis of proteins. However, by performing digestion in microdroplets generated from electrosonic spray, the reaction could be accelerated to a timescale lower than milliseconds. Here, we describe a simple and rapid online digestion platform named online microdroplet-assisted enzymatic digestion (MAED). It involves the integration of intact protein separation with enzymatic digestion in microdroplets. Via online MAED, various protein standards, including an antibody standard, were characterized in a bottom-up manner without prior digestion, and high sequence coverages were obtained. We further extended the application of online MAED to a more complex sample, mouse brain extract, where protein identifications were successfully yielded. Compared with the conventional bottom-up approach, a more comprehensive characterization could be obtained particularly for low molecular weight proteins. In short, we provide a rapid and alternative bottom-up analysis in a top-down fashion as well as a new possibility for microdroplet chemistry.en[SDGs]SDG3journal article10.1039/d3cc03509c377898212-s2.0-85174545067https://api.elsevier.com/content/abstract/scopus_id/85174545067