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

Abstract

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.

Figure 1

Schematic representation of the multi-dimensional HPLC platform for affinity purification of clustrein. Columns were independently switched on- or off-line for sample binding, column washing, and elution purposes.

Figure 2

(a) SDS-PAGE analysis of immune-affinity enriched clusterin from control plasma and (b) Commassie stained 2-DE separation of affinity purified clusterin from 150 ug plasma, first dimension: IEF pH range 4 to 7 and second dimension: 10% SDS-PAGE. The spots highlighted represent different isoforms of clusterin confirmed by LC-MS analysis (data shown in table.1). clusterin spots were excised and pooled for glycomic analysis.

Figure 3

Representative UPLC-HILIC chromatogram of clusterin N-glycan displaying 16 integrated peaks used for multivariate statistical analysis and Wilcoxon signed rank test

Figure 4

PCA plot generated using multivariate statistical analysis of HILIC-fluorescence glycan profiles of clusterin isolated from RCC patients plasma before ( ) and after (◆) surgery showing Pre samples clustering together and controls (post surgery) dividing into a group of 4 and a group of 2.

Figure 5

ELISA measurement of the level of clusterin in plasma of RCC patients before and after surgery.