In-House Packed Porous Graphitic Carbon Columns for Liquid Chromatography-Mass Spectrometry Analysis of N-Glycans

Protein glycosylation is a common post-translational modification that modulates biological processes such as the immune response and protein trafficking. Altered glycosylation profiles are associated with cancer and inflammatory diseases, as well as impacting the efficacy of therapeutic monoclonal antibodies. Consisting of oligosaccharides attached to asparagine residues, enzymatically released N-linked glycans are analytically challenging due to the diversity of isomeric structures that exist. A commonly used technique for quantitative N-glycan analysis is liquid chromatography-mass spectrometry (LC-MS), which performs glycan separation and characterization. Although many reversed and normal stationary phases have been utilized for the separation of N-glycans, porous graphitic carbon (PGC) chromatography has become desirable because of its higher resolving capability, but is difficult to implement in a robust and reproducible manner. Herein, we demonstrate the analytical properties of a 15 cm fused silica capillary (75 mu m i.d., 360 mu m o.d.) packed in-house with Hypercarb PGC (3 mu m) coupled to an Agilent 6550 Q-TOF mass spectrometer for N-glycan analysis in positive ion mode. In repeatability and intermediate precision measurements conducted on released N-glycans from a glycoprotein standard mixture, the majority of N-glycans reported low coefficients of variation with respect to retention times (<= 4.2%) and peak areas (<= 14.4%). N-glycans released from complex samples were also examined by PGC LC-MS. A total of 120 N-glycan structural and compositional isomers were obtained from formalin-fixed paraffin-embedded ovarian cancer tissue sections. Finally, a comparison between early- and late-stage formalin-fixed paraffin-embedded ovarian cancer tissues revealed qualitative changes in the alpha 2,3- and alpha 2,6-sialic acid linkage of a fucosylated bi-antennary complex N-glycan. Although the alpha 2,3-linkage was predominant in late-stage ovarian cancer, the alternate alpha 2,6-linkage was more prevalent in early-stage ovarian cancer. This study establishes the utility of in-house packed PGC columns for the robust and reproducible LC-MS analysis of N-glycans.

Automated summary

Protein glycosylation plays a crucial role in biological processes, with liquid chromatography-mass spectrometry being a commonly used technique for N-glycan analysis. The study demonstrates the utility of in-house packed PGC columns for robust and reproducible LC-MS analysis of N-glycans, highlighting the importance of studying N-glycan structures in ovarian cancer tissues.