Hybrid Capillary/Microfluidic System for Comprehensive Online Liquid Chromatography-Capillary Electrophoresis-Electrospray Ionization-Mass Spectrometry
journal contributionposted on 16.04.2013, 00:00 by J. Scott Mellors, William A. Black, Andrew G. Chambers, Jason A. Starkey, Nathan A. Lacher, J. Michael Ramsey
A hybrid multidimensional separation system was made by coupling capillary liquid chromatography (LC) to a microfluidic device. The microfluidic device integrated flow splitting, capillary electrophoresis (CE), electroosmotic pumping, and electrospray ionization (ESI) emitter functional elements. The system was used with a time-of-flight mass spectrometer for comprehensive online LC-CE-MS of proteolytic digests. Analysis of a complex mixture of peptides yielded a peak capacity of approximately 1400 in 50 min. Three replicate runs demonstrated mean reproducibility for LC retention and CE migration times of 0.32% and 0.75% relative standard deviation (RSD), respectively. The same LC-CE-MS method was also used to characterize the N-linked glycosylation of a monoclonal antibody. Glycopeptides from two different N-linked glycosylation sites were separated from all other tryptic peptides and identified using MS data. The relative amounts of each glycoform and total site occupancy were quantified using LC-CE-MS data.
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SpectrometryAelectrosprayHybridmicrofluidic deviceOnlineglycoformdatadigestretentionelectrophoresisGlycopeptidecapacitychromatographyminCE migration timesComprehensiveoccupancyreproducibilityCapillarymethoddeviationmixturepeptidesplittingionizationseparationspectrometercapillaryemitterESILCglycosylationRSDantibodyflowanalysistrypticelectroosmoticMSsite