Simple and Rapid Proteomic Analysis by Protease-Immobilized Microreactors

Proteomics is the large-scale study of proteins, particularly their stuructures and functions. One of the most important point is to develop efficient and rapid approachehes to identify the target proteins. Peptide mass mapping and tandem mass spectrometry (MS/MS)-based peptide sequencing are key methods in current protein identification for proteomic studies. Proteins are usually digested into peptides that are subsequently analyzed by MS. Therefore, proteolysis by sequence-specific proteases is the key step for positive sequencing in proteomic analysis integrated with MS (Aebersold & Mann, 2003). The conventional method of in-solution digestion by proteases is a time-consuming procedure (overnight at 37 C). The substrate/protease ratio must be kept high (generally > 50) in order to prevent excessive sample contamination by the protease and its auto-digested products. But this leads to a relatively slow digestion. In addition, obtaining reliable peptide maps and meaningful sequence data by MS analysis requires not only the separation of the digested peptides but also strictly defined proteolysis conditions (Domon & Aebersold, 2006; Witze et al., 2007). In addition, the ionization efficiency of the digested fragments including a modified peptide such as a phosphopeptide is dependent on peptide-size or peptidesequence, which directly correlates with sequence coverage of the target protein by MS analysis. Furthermore, peptide recovery from in-solution digestion is highly dependent on the structural properties of the target proteins because proteins with rigid structures, e.g. by disulfide bonds tend to be resistant to complete digestion. In fact, the typical preparation of a sample for proteolysis includes denaturation, reduction of disulfide bonds, and alkylation procedures lead to a decrease the conformational stability. It is obvious that insufficient sequence coverage could compromise the accuracy of proteome characterization. Therefore, it is important to develop novel digestion methods to achieve a highly efficient proteolysis for MS-based peptide mapping (Park & Russell, 2000; Slysz et al., 2006). A microreactor is a suitable reaction system for handling small-volume samples in a microchannel to perform chemical or enzymatic reactions. Enzyme-immobilized