Development of a multiplexed microfluidic proteomic reactor and its application for studying protein-protein interactions
| Autor: | Daniel Figeys, Ruijun Tian, Xuyen Dai Hoa, John Paul Pezacki, Jean-Philippe Lambert, Teodor Veres |
|---|---|
| Rok vydání: | 2011 |
| Předmět: |
Proteomics
Multiple samples Molecular biology Polymers Limit of detection Protein complexes Microfluidics Thermoplastic materials Cyclic olefin copolymer Mass Spectrometry Analytical Chemistry Histones chemistry.chemical_compound Protein purification Protein Interaction Mapping Gel electrophoresis Histone variants instrumentation Protein digestion protein domain Immunopurification methodology Wild types Microfluidic Analytical Techniques Manual processing Reinforced plastics Complexation Protein separations Electrophoresis Protein samples Chromatin Immunoprecipitation Error prones Saccharomyces cerevisiae Proteins Immunoprecipitation Protein-protein interactions polymer histone Mass spectrometry chemistry Yeast strain Olefins Replication techniques Saccharomyces cerevisiae protein Immunoprecipitations Protein Interaction Domains and Motifs Styrene Chromatography Proteins Htz1 protein S cerevisiae Yeast protein analysis Parallel analysis microfluidic analysis Hot-embossing Gels Cyclic Olefin Copolymers |
| Zdroj: | Analytical chemistry. 83(11) |
| ISSN: | 1520-6882 |
| Popis: | 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. © 2011 American Chemical Society. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|