Multidimensional liquid chromatography platform for profiling alterations of clusterin N-glycosylation in the plasma of patients with renal cell carcinoma

Identification of potential changes in the glycosylation of existing cancer biomarkers can result in a higher level of diagnostic sensitivity and specificity. Clusterin (Apolipoprotein J) has been implicated in renal cell carcinoma (RCC) and other types of malignancy as potential biomarker. In the present work, an automated multi-dimensional HPLC platform enabling high throughput affinity enrichment of clusterin from plasma samples was developed. Integrated with two dimensional gel electrophoresis, high purity clusterin in microgram quantities suitable for glycan characterization was isolated. The analytical platform was applied to study clusterin glycosylation in a small group of RCC patients before and after nephrectopy as a pilot study to evaluate the performance of the platform. A statistically significant decrease was observed in the levels of a bi-antennary digalactosyl disialylated (A2G2S(3)2) glycans while the levels of a core fucosylated bi-antennary digalactosyl disialylated glycan (FA2G2S(6)2) and a tri-antennary trigalactosyl disialylated glycan (A3G3S(6)2) were increased in the post-surgery plasma samples.

► Multidimensional HPLC platform for high efficiency enrichment of clusterin from plasma.
► Combined with 2-DE, high purity clusterin in μg quantity needed for glycomic analysis was obtained.
► Application of the platform to N-glycomic characterization of enriched clusterin described.
► Alterations in clusterin glycosylation associated with the presence of the cancer were observed.
► Observed alterations in glycosylation may serve as potential markers with further verification.