Hybrid capillary/microfluidic system for comprehensive online liquid chromatography-capillary electrophoresis-electrospray ionization-mass spectrometry.

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.

[1]  H. Desaire,et al.  Glycopeptide analysis by mass spectrometry. , 2008, The Analyst.

[2]  J. Ramsey,et al.  Monolithic integration of two-dimensional liquid chromatography-capillary electrophoresis and electrospray ionization on a microfluidic device. , 2011, Analytical chemistry.

[3]  O. Krokhin.  Peptide retention prediction in reversed-phase chromatography: proteomic applications , 2012, Expert review of proteomics.

[4]  X. Zhang,et al.  Comprehensive two‐dimensional separations based on capillary high‐performance liquid chromatography and microchip electrophoresis , 2003, Electrophoresis.

[5]  J Michael Ramsey,et al.  High-efficiency, two-dimensional separations of protein digests on microfluidic devices. , 2003, Analytical chemistry.

[6]  Alain Van Dorsselaer,et al.  The way forward, enhanced characterization of therapeutic antibody glycosylation: comparison of three level mass spectrometry-based strategies. , 2008, Journal of chromatography. B, Analytical technologies in the biomedical and life sciences.

[7]  K. Markides,et al.  A simplified multidimensional approach for analysis of complex biological samples: on-line LC-CE-MS. , 2006, The Analyst.

[8]  J. Michael Ramsey,et al.  Effects of injection schemes and column geometry on the performance of microchip electrophoresis devices , 1994 .

[9]  N Gottschlich,et al.  Two-dimensional electrochromatography/capillary electrophoresis on a microchip. , 2001, Analytical chemistry.

[10]  Lloyd M Smith,et al.  Integrated microfluidic device for automated single cell analysis using electrophoretic separation and electrospray ionization mass spectrometry. , 2010, Analytical chemistry.

[11]  J. Jorgenson,et al.  Comprehensive three-dimensional separation of peptides using size exclusion chromatography/reversed phase liquid chromatography/optically gated capillary zone electrophoresis. , 1995, Analytical chemistry.

[12]  S. Reichenbach,et al.  Smart templates for peak pattern matching with comprehensive two-dimensional liquid chromatography. , 2009, Journal of chromatography. A.

[13]  J. Jorgenson,et al.  Automated instrumentation for comprehensive two-dimensional high-performance liquid chromatography/capillary zone electrophoresis , 1990 .

[14]  J. Jorgenson,et al.  A Transparent Flow Gating Interface for the Coupling of Microcolumn LC with CZE in a Comprehensive Two-Dimensional System , 1993 .

[15]  G. Whitesides.  The origins and the future of microfluidics , 2006, Nature.

[16]  G. Opiteck,et al.  Comprehensive on-line RPLC-CZE-MS of peptides , 1997 .

[17]  J. Jorgenson,et al.  Two-dimensional liquid chromatography-capillary electrophoresis techniques for analysis of proteins and peptides. , 1996, Methods in enzymology.

[18]  S. K. Griffiths,et al.  Low-dispersion turns and junctions for microchannel systems. , 2001, Analytical chemistry.

[19]  J. Jorgenson,et al.  Two‐dimensional separations of peptides and proteins by comprehensive liquid chromatography‐capillary electrophoresis , 1993, Electrophoresis.

[20]  J. Ramsey,et al.  Fully integrated glass microfluidic device for performing high-efficiency capillary electrophoresis and electrospray ionization mass spectrometry. , 2008, Analytical chemistry.

[21]  J. Jorgenson,et al.  Rapid comprehensive two-dimensional separations of peptides via RPLC-optically gated capillary zone electrophoresis. , 1995, Analytical chemistry.

[22]  Hong Chen,et al.  Two‐dimensional protein separation in microfluidic devices , 2009, Electrophoresis.

[23]  Reb J. Russell,et al.  Characterization of glycosylation sites for a recombinant IgG1 monoclonal antibody and a CTLA4-Ig fusion protein by liquid chromatography-mass spectrometry peptide mapping. , 2011, Journal of chromatography. A.

[24]  A. Lim,et al.  Glycosylation profiling of a therapeutic recombinant monoclonal antibody with two N-linked glycosylation sites using liquid chromatography coupled to a hybrid quadrupole time-of-flight mass spectrometer. , 2008, Analytical biochemistry.

[25]  Steven A Soper,et al.  Two-dimensional electrophoretic separation of proteins using poly(methyl methacrylate) microchips. , 2006, Analytical chemistry.

[26]  K. Markides,et al.  Development of a poly(dimethylsiloxane) interface for on-line capillary column liquid chromatography-capillary electrophoresis coupled to sheathless electrospray ionization time-of-flight mass spectrometry. , 2003, Analytical chemistry.

[27]  P. Marriott,et al.  Retention indices in comprehensive two-dimensional gas chromatography , 2011, Analytical and bioanalytical chemistry.

[28]  Lukas Käll,et al.  Training, selection, and robust calibration of retention time models for targeted proteomics. , 2010, Journal of proteome research.

[29]  Samuel Tia,et al.  On-chip technologies for multidimensional separations. , 2009, Lab on a chip.

[30]  J. Jorgenson,et al.  Two-dimensional protein separation by microcolumn size-exclusion chromatography-capillary zone electrophoresis , 1993 .

[31]  J. Sweedler,et al.  Multidimensional separation of chiral amino acid mixtures in a multilayered three-dimensional hybrid microfluidic/nanofluidic device. , 2009, Analytical chemistry.

[32]  A comparison of tryptic digests of bovine and equine cytochrome C by comprehensive reversed‐phase HPLC–CE , 1990 .

[33]  J. Ramsey,et al.  A microfabricated fluidic device for performing two-dimensional liquid-phase separations. , 2000, Analytical chemistry.