Online Integration of Multiple Sample Pretreatment Steps Involving Denaturation, Reduction, and Digestion with Microflow Reversed-Phase Liquid Chromatography−Electrospray Ionization Tandem Mass Spectrometry for High-Throughput Proteome Profiling

Abstract

A facile integrated platform for proteome profiling was established, in which native proteins were online denatured and reduced within a heater, digested with an immobilized trypsin microreactor, and analyzed by microflow reversed-phase liquid chromatography with electrospray ionization tandem mass spectrometry (microRPLC-ESI-MS/MS). In comparison to the traditional off-line urea denaturation protocol, even more unique peptides were obtained by online heating in triplicate (14 +/- 2 vs 11 +/- 2 for myoglobin and 16 vs 12 +/- 1 for BSA) within a significantly shortened pretreatment time of approximately 3.5 min (including 1 min of thermal denaturation and reduction and approximately 2.5 min of microreactor digestion). Moreover, proteins with concentrations ranging from 50 ng/mL (approximately 6 fmol) to 1 mg/mL (approximately 120 pmol) were positively identified by the online system. Such a platform was further successfully applied for analyzing the soluble fraction of mouse liver extract. Of all the 367 proteins identified from samples pretreated by the urea protocol and online heating, approximately 40% were overlapped, showing the partial complementation of both approaches. All these results demonstrate that the online integrated platform is of great promise for high-throughput proteome profiling and improved identification capacity for low-abundance proteins with a minute sample amount.