Multidigestion in continuous flow tandem protease-immobilized microreactors for proteomic analysis.

Proteolysis by sequence-specific proteases is the key step for positive sequencing in proteomic studies integrated with mass spectrometry (MS). The conventional method of in-solution digestion of protein is a time-consuming procedure and has limited sensitivity. In this study, we report a simple and rapid system for the analysis of protein sequence and protein posttranslational modification by multienzymatic reaction in a continuous flow using the enzyme (trypsin, chymotrypsin, or alkaline phosphatase)-immobilized microreactor. The feasibility and performance of the single microreactor and tandem microreactors that were connected by the different microreactors were determined by the digestion of nonphosphoprotein (cytochrome c) and phosphoproteins (β-casein and pepsin A). The single microreactor showed rapid digestion compared with that of in-solution digestions. Multiple digestion by the tandem microreactors showed higher sequence coverage compared with that by in-solution or the single microreactor. Moreover, the tandem microreactor that was made by using the combination of protease-immobilized microreactor and phosphatase-immobilized microreactor showed the capability for phosphorylation site analysis in phosphoproteins without the use of any enrichment strategies or radioisotope labeling techniques. This approach provides a strategy that can be applied to various types of linking microreactor-based multienzymatic reaction systems for proteomic analysis.