title: Development of a multiplexed microfluidic proteomic reactor and its application for studying protein-protein interactions.
abstract: Mass spectrometry-based proteomics techniques have been very successful for the identification and study of protein-protein interactions. Typically, immunopurification of protein complexes is conducted, followed by protein separation by gel electrophoresis and in-gel protein digestion, and finally, mass spectrometry is performed to identify the interacting partners. However, the manual processing of the samples is time-consuming and error-prone. Here, we developed a polymer-based microfluidic proteomic reactor aimed at the parallel analysis of minute amounts of protein samples obtained from immunoprecipitation. The design of the proteomic reactor allows for the simultaneous processing of multiple samples on the same devices. Each proteomic reactor on the device consists of SCX beads packed and restricted into a 1 cm microchannel by two integrated pillar frits. The device is fabricated using a combination of low-cost hard cyclic olefin copolymer thermoplastic and elastomeric thermoplastic materials (styrene/(ethylene/butylenes)/styrene) using rapid hot-embossing replication techniques with a polymer-based stamp. Three immunopurified protein samples are simultaneously captured, reduced, alkylated, and digested on the device within 2-3 h instead of the days required for the conventional protein-protein interaction studies. The limit of detection of the microfluidic proteomic reactor was shown to be lower than 2 ng of protein. Furthermore, the application of the microfluidic proteomic reactor was demonstrated for the simultaneous processing of the interactome of the histone variant Htz1 in wild-type yeast and in a swr1Δ yeast strain compared to an untagged control using a novel three-channel microfluidic proteomic reactor.
authors.affiliation: Ottawa Institute of Systems Biology, National Research Council, Boucherville, QC, Canada J4B 6Y4.
authors.forename: Ruijun
authors.initials: R
authors.lastname: Tian
authors.forename: Xuyen Dai
authors.initials: XD
authors.lastname: Hoa
authors.forename: Jean-Philippe
authors.initials: JP
authors.lastname: Lambert
authors.forename: John Paul
authors.initials: JP
authors.lastname: Pezacki
authors.forename: Teodor
authors.initials: T
authors.lastname: Veres
authors.forename: Daniel
authors.initials: D
authors.lastname: Figeys
mesh_terms: D047369:Chromatin Immunoprecipitation; D006657:Histones / Q000737:chemistry*; D013058:Mass Spectrometry / Q000379:methods; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation / Q000379:methods*; D011108:Polymers / Q000737:chemistry; D054730:Protein Interaction Domains and Motifs; D025941:Protein Interaction Mapping / Q000379:methods*; D040901:Proteomics / Q000295:instrumentation / Q000379:methods*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry*
chemical_list: D006657:Histones; C493530:Htz1 protein, S cerevisiae; D011108:Polymers; D029701:Saccharomyces cerevisiae Proteins