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

TLDR

Glycosylation, a diverse post‑translational modification, is crucial for protein function and its alterations, including in cancer, are being explored as serum biomarker candidates. The study demonstrates that ultra‑performance liquid chromatography on a sub‑2 µm HILIC phase markedly improves efficiency, selectivity, and speed for profiling fluorescently labeled N‑linked glycans, enabling detection of cancer‑associated changes in the serum N‑glycome of stomach adenocarcinoma patients. The authors evaluated glycosylation on four abundant serum proteins—IgG, haptoglobin, transferrin, and α1‑acid glycoprotein—using affinity purification or two‑dimensional electrophoresis to assess their potential as markers of stomach cancer progression. They found increased sialylation on haptoglobin, transferrin, and α1‑acid glycoprotein, and on IgG, higher core‑fucosylated biantennary glycans with lower monogalactosylated core‑fucosylated biantennary glycans correlated with disease progression, while the sub‑2 µm HILIC phase provided rapid, highly selective separation of complex serum N‑glycan mixtures.

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.