title: Hybrid capillary/microfluidic system for comprehensive online liquid chromatography-capillary electrophoresis-electrospray ionization-mass spectrometry.
abstract: 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.
authors.affiliation: Department of Chemistry, University of North Carolina, Chapel Hill, North Carolina 27599, United States.
authors.forename: J Scott
authors.initials: JS
authors.lastname: Mellors
authors.forename: William A
authors.initials: WA
authors.lastname: Black
authors.forename: Andrew G
authors.initials: AG
authors.lastname: Chambers
authors.forename: Jason A
authors.initials: JA
authors.lastname: Starkey
authors.forename: Nathan A
authors.initials: NA
authors.lastname: Lacher
authors.forename: J Michael
authors.initials: JM
authors.lastname: Ramsey
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry; D002417:Cattle; D002853:Chromatography, Liquid / Q000295:instrumentation*; D019075:Electrophoresis, Capillary / Q000295:instrumentation*; D006020:Glycopeptides / Q000737:chemistry; D006031:Glycosylation; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation*; D010446:Peptide Fragments / Q000032:analysis*; D011134:Polysaccharides / Q000032:analysis*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012710:Serum Albumin, Bovine / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization / Q000295:instrumentation*; D014357:Trypsin
chemical_list: D000911:Antibodies, Monoclonal; D006020:Glycopeptides; D010446:Peptide Fragments; D011134:Polysaccharides; D012710:Serum Albumin, Bovine; D014357:Trypsin