Abstract
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 µm i.d., 360 µm o.d.) packed in-house with Hypercarb PGC (3 µm) 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 α2,3- and α2,6-sialic acid linkage of a fucosylated bi-antennary complex N-glycan. Although the α2,3-linkage was predominant in late-stage ovarian cancer, the alternate α2,6-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.
Highlights
In humans, the glycosylation machinery responsible for the orchestrated glycosylation of proteins is encoded by approximately 700 human genes (Schjoldager et al, 2020)
We evaluated 10 mM ammonium bicarbonate (Jensen et al, 2012) and 0.04% ammonium hydroxide (Thomsson et al, 2010) as they were successfully used in negative ion mode porous graphitic carbon (PGC) liquid chromatography-mass spectrometry (LC-mass spectrometry (MS))
The chromatographical capabilities of the 15 cm fused silica column packed with 3 μm Hypercarb was evaluated using N-glycans released from a glycoprotein standard mixture using a 70 min gradient
Summary
The glycosylation machinery responsible for the orchestrated glycosylation of proteins is encoded by approximately 700 human genes (Schjoldager et al, 2020). Glycosylation has the ability to modify the physicochemical properties and biological functions of proteins (Lis and Sharon, 1993), which influences many cellular processes such as the immune response (Rudd et al, 2001) and protein trafficking (Varki, 2017). The development of analytical tools that discriminate glycosylation profiles is important from a clinical perspective and for characterizing the role of glycosylation in the efficacy and safety of protein therapeutics (Liu, 2015). Chemical derivatization of glycans is often employed following PNGase F digestion to aid their detection in LC-MS (Ruhaak et al, 2010). Many methodologies exist to obtain both qualitative and quantitative glycomics data, which reflects the immense diversity and hydrophilic nature of N-glycans
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