IgG N-glycans as potential biomarkers for determining galactose tolerance in Classical Galactosaemia.

N-glycan processing and assembly defects have been demonstrated in untreated and partially treated patients with Classical Galactosaemia. These defects may contribute to the ongoing pathophysiology of this disease. The aim of this study was to develop an informative method of studying differential galactose tolerance levels and diet control in individuals with Galactosaemia, compared to the standard biochemical markers. Ten Galactosaemia adults with normal intellectual outcomes were analyzed in the study. Five subjects followed galactose liberalization, increments of 300 mg to 4000 mg/day over 16 weeks, and were compared to five adult Galactosaemia controls on a galactose restricted diet. All study subjects underwent clinical and biochemical monitoring of red blood cell galactose-1-phosphate (RBC Gal-1-P) and urinary galactitol levels. Serum N-glycans were isolated and analyzed by normal phase high-performance liquid chromatography (NP-HPLC) with galactosylation of IgG used as a specific biomarker of galactose tolerance. IgG N-glycan profiles showed consistent individual alterations in response to diet liberalization. The individual profiles were improved for all, but one study subject, at a galactose intake of 1000 mg/day, with decreases in agalactosylated (G0) and increases in digalactosylated (G2) N-glycans. We conclude that IgG N-glycan profiling is an improved method of monitoring variable galactosylation and determining individual galactose tolerance in Galactosaemia compared to the standard methods.

Levels of specific serum N-glycans identify breast cancer patients with higher circulating tumor cell counts.

BACKGROUND: Metastatic breast cancer (MBC) is currently an incurable condition that is primarily treated with palliative measures. Isolation of circulating tumor cells (CTCs) from the peripheral blood of these patients provides a predictive prognostic indicator, independent of the type of therapy, site of occurrence and biological characteristics of the primary disease. It has been well established that glycosylation processing pathways are disturbed in cancer, leading to alterations in the glycan content of glycoproteins. MATERIALS AND METHODS: The bi-, tri- and tetraantennary glycans containing sialyl Lewis x (sLe(x)) epitopes (A2F1G1, A3F1G1, A4F1G1 and A4F2G2) were quantified using normal phase high-performance liquid chromatography in combination with exoglycosidase array digestions in the glycan pools released from sera of 27 patients with advanced breast cancer (16 with CTCs <5/7.5 ml and 11 with CTCs ≥5/7.5 ml) and 13 healthy women. RESULTS: The levels of all these glycans were significantly higher in patients with CTCs ≥5/7.5 ml compared with patients with CTCs <5/7.5 ml. CONCLUSIONS: As high levels of glycans containing sLe(x) epitopes were associated with CTCs, their measurement may provide a new noninvasive approach for determining prognosis in women with MBC.

Automated, high-throughput serum glycoprofiling platform.

Complex carbohydrates are rapidly becoming excellent biomarker candidates because of their high sensitivity to pathological changes. However, the discovery of clinical glycobiomarkers has been slow, due to the scarcity of high-throughput glycoanalytical workflows that allow rapid glycoprofiling of large clinical sample sets. To generate high-quality quantitative glycomics data in a high-throughput fashion, we have developed a robotized platform for rapid serum-based N-glycan sample preparation. The sample preparation workflow features a fully automated, rapid glycoprotein denaturation followed by sequential enzymatic glycan release, glycan purification on solid-supported hydrazide and fluorescent labelling. This allows accurate glycan quantitation by ultra-high performance liquid chromatography (UPLC). The sample preparation workflow was automated using an eight-channel Hamilton Robotics liquid handling workstation, allowing the preparation of almost 100 samples in 14 hours with excellent reproducibility and thus should greatly facilitate serum-based glyco-biomarker discovery.