A nanoHILIC-MS Platform for Separation and Characterization of Glycoproteins

A nanoHILIC-MS platform was developed to provide a means for top-down and middle-up analysis of glycoproteins. A mechanistic approach was taken in evaluating characteristics of the stationary phase of the chromatography column. Results from this evaluation informed the selection of a polyacrylamide brush layer-based bonded phase, which was applied to nonporous silica nanoparticles. Fluorescence microscopy was used for direct analysis of labeled proteins during nanoHILIC separations, with the platform subsequently adapted for mass spectrometry-based detection of intact and semi-intact glycoproteins. It was found that the standard technique of linking a hollow needle emitter to the capillary chromatography column for mass spectrometry-based detection yielded an unacceptable level of band broadening, necessitating the development of a column with an integrated needle emitter. The resulting nanoHILIC-MS method yielded peak widths as sharp as 3.5 seconds, enabling ultra-sensitive detection and identification of 28 glycoforms of an IgG1κ monoclonal antibody standard.