Ultra Performance Liquid Chromatographic Profiling of Serum N-Glycans for Fast and Efficient Identification of Cancer Associated Alterations in Glycosylation

Abstract

Glycosylation is a diverse but critically important post-translational modification that modulates the physical, chemical and biological properties of proteins. Alterations in glycosylation have been noted in a number of diseases including cancer. The discovery of alterations in the glycosylation of serum glycoproteins which may offer potential as biomarkers is attracting considerable research interest. In the current study, the significant improvements in efficiency, selectivity, and analysis speed offered by ultra performance liquid chromatography (UPLC) profiling of fluorescently labeled N-linked oligosaccharides on a recently introduced sub-2 μm hydrophilic interaction (HILIC) based stationary phase are demonstrated to identify cancer associated alterations in the serum N-glycome of patients bearing stomach adenocarcinoma. The contribution of the glycosylation present on four highly abundant serum proteins namely, IgG, haptoglobin, transferrin, and α1-acid glycoprotein was evaluated. Alterations in the glycosylation present on these four proteins isolated from the pathologically staged cancer serum using either affinity purification or two-dimensional electrophoresis were then investigated as possible markers for stomach cancer progression. In agreement with previous reports, an increase in sialylation was observed on haptoglobin, transferrin, and α1-acid glycoprotein in the cancerous state. Increased levels of core fucosylated biantennary glycans and decreased levels of monogalactosylated core fucosylated biantennary glycans were present on IgG with increasing disease progression. The speed and selectivity offered by the sub-2 μm HILIC phase make it ideal for rapid yet highly efficient separation of complex oligosaccharide mixtures such as that present in the serum N-glycome.

Comparison of separation techniques for the elucidation of IgG N-glycans pooled from healthy mammalian species

“…3). The major glycans from bovine IgG were fucosylated, with over 80% of glycans consisting of a combination of FA2 (11.9%), FA2G1 (FA2 [6]G1 and FA2 [3]G1; 9.1% and 19.6%, respectively), and FA2G2 (24.4%). There were roughly 20% bisected biantennary glycans also identified within the profile.…”

Comparison of methods for the analysis of therapeutic immunoglobulin G Fc-glycosylation profiles—Part 1: Separation-based methods

“…20 A wide range of state-of-the-art analytical methods to monitor Fc-glycosylation is available. In principle the methods can be sub-divided into 3 categories: 3,[21][22][23] (1) Analysis of the IgG molecule with electrospray ionization mass spectrometry (ESI-MS)-either on the intact molecule after reduction of disulphide bonds, or after a limited digestion with a proteolytic enzyme and deduction of the overall glycan composition; [24][25][26] (2) Enzymatic release of the Fc glycans and measurement with mass spectrometric methods, by HPLC with pulsed amperometric detection or by capillary electrophoresis (CE)/ HPLC-based methods after fluorescent labeling; 27,28 and (3) Proteolytic cleavage of the IgG molecule and analysis of the glycopeptides with matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) or electrospray ionization-mass spectrometry ESI-MS. [29][30][31][32] Comparisons of different methods for analysis of IgG Fc-glycosylation have been reported, but these studies included a limited number of methods or compared mainly mass spectrometry-based methods. [33][34][35][36][37][38] Thus, a thorough comparison of different methods for glycoanalysis is still lacking.…”

Recent Developments in Liquid Chromatography and Capillary Electrophoresis for the Analysis of Glycoprotein Glycans

“…[115][116][117] HILIC tolerates neutral and acidic glycans, regardless of whether they are labeled with fluorescent tags. 118 Typical equilibration conditions in the HILIC HPLC of mono-and oligosaccharides and glycopeptides are 10 -25% water in acetonitrile with a low concentration of acid or salt (mostly below 100 mM).…”