title: Integrated microfluidic device for mass spectrometry-based proteomics and its application to biomarker discovery programs.
abstract: The present investigation describes the analytical performances of a microfluidic device comprising an enrichment column, a reversed-phase separation channel, and a nanoelectrospray emitter embedded altogether in polyimide layers. This configuration minimizes transfer lines and connections and reduces postcolumn peak broadening and dead volumes. This compact and versatile modular nanoLC-chip system was interfaced to both ion trap and time-of-flight mass spectrometers, and its analytical potentials were evaluated in the context of proteomics applications. The figures of merit of this system in terms of peak capacity, reproducibility, sensitivity, and linear dynamic range of peptide detection were determined using tryptic digests of complex protein extracts including albumin- and immunoglobulin-depleted rat plasma samples. The analysis of peak profiles for more than 600 peptide ions reproducibly detected across replicate nanoLC-chip-MS runs (n = 10) indicated that this system provided good reproducibility of retention time and peak intensity with RSD values of less than 0.5 and 9.1%, respectively. Variation in peptide abundance as low as 2-fold changes was identified for spiked tryptic digests present at levels of 2-5 fmol in plasma samples. Sensitivity measurements were performed on dilution series of protein digests spiked into rat plasma samples and provided a detection limit of 1-5 fmol. The modular concept of the microfluidic systems also facilitated the integration of two-dimensional chromatography (strong cation exchange/C18) thereby increasing the sample loading and selectivity of the nanoLC-chip-MS system. The application of this integrated device was evaluated for complex rat plasma samples to compare the number of protein identifications obtained using one- and two-dimensional nanoLC-chip-MS/MS.
authors.affiliation: Department of Chemistry, Université de Montréal, Montréal, Canada, Caprion Pharmaceuticals, Montréal, Canada, Institute for Research in Immunology and Cancer, Université de Montréal, Montreal, Canada.
authors.forename: Marie-Helene
authors.initials: MH
authors.lastname: Fortier
authors.forename: Eric
authors.initials: E
authors.lastname: Bonneil
authors.forename: Paul
authors.initials: P
authors.lastname: Goodley
authors.forename: Pierre
authors.initials: P
authors.lastname: Thibault
mesh_terms: D015415:Biomarkers; D001798:Blood Proteins / Q000032:analysis*; D002853:Chromatography, Liquid; D013058:Mass Spectrometry / Q000379:methods*; D046210:Microfluidic Analytical Techniques; D040901:Proteomics; D015203:Reproducibility of Results
chemical_list: D015415:Biomarkers; D001798:Blood Proteins