title: Integrated microfluidic devices with enhanced separation performance: application to phosphoproteome analyses of differentiated cell model systems.
abstract: This work reports on the application of a microfluidic device integrating nanoscale LC to nanoelectrospray MS (nano-LC-chip-MS) for the analysis of complex protein digests. Peak profile analyses of more than 700 peptide ions, reproducibly detected across replicate nano-LC-chip-MS runs (n = 5), indicated that the system provided RSD values of 0.24% on retention time, +/- 30 ppm on m/z measurement and +/- 30% variation on intensity over three orders of magnitude. RP adsorbant media with different alkyl chains and particle size packed in both trapping and separation channels were investigated to improve the chromatographic performance of this system. A two-fold improvement in chromatographic peak capacity was achieved using microfluidic devices comprising a 5 mircrom C3 trap with 2.5 microm C18 trap separation channel compared to the traditional 5 microm C18 stationary phase. Enhanced sample selectivity for the identification of phosphopeptides was obtained by combining immobilized metal affinity media prior to peptide separation on the RP microfluidic device. This system was evaluated in the context of differential phosphoproteome analyses to identify changes in signaling events and protein expression of human monocytes following the administration of phorbol ester.
authors.affiliation: Institute for Research in Immunology and Cancer, Université de Montreal, Quebec, Canada.
authors.forename: Mihaela
authors.initials: M
authors.lastname: Ghitun
authors.forename: Eric
authors.initials: E
authors.lastname: Bonneil
authors.forename: Marie-Helene
authors.initials: MH
authors.lastname: Fortier
authors.forename: Hongfeng
authors.initials: H
authors.lastname: Yin
authors.forename: Kevin
authors.initials: K
authors.lastname: Killeen
authors.forename: Pierre
authors.initials: P
authors.lastname: Thibault
mesh_terms: D000818:Animals; D002460:Cell Line; D002853:Chromatography, Liquid / Q000379:methods; D006801:Humans; D051379:Mice; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation* / Q000379:methods; D036103:Nanotechnology; D010750:Phosphoproteins / Q000302:isolation & purification*; D020543:Proteome / Q000302:isolation & purification; D040901:Proteomics / Q000295:instrumentation* / Q000379:methods; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D020298:U937 Cells
chemical_list: D010750:Phosphoproteins; D020543:Proteome; D013755:Tetradecanoylphorbol Acetate