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

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 (μRPLC-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 ∼3.5 min (including 1 min of thermal denaturation and reduction and ∼2.5 min of microreactor digestion). Moreover, proteins with concentrations ranging from 50 ng/mL (∼6 fmol) to 1 mg/mL (∼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, ∼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.