Real-time bottom-up characterization of protein mixtures enabled by online microdroplet-assisted enzymatic digestion (MAED)

Abstract

Enzymatic 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.