Integrated workflow for urinary prostate specific antigen N-glycosylation analysis using sdAb partitioning and downstream capillary electrophoresis separation.

Prostate cancer represents the second highest malignancy rate in men in all cancer diagnoses worldwide. The development and progression of prostate cancer is not completely understood yet at molecular level, but it has been reported that changes in the N-glycosylation of prostate-specific antigen (PSA) occur during tumor genesis. In this paper we report on the development and implementation of a high-throughput capillary electrophoresis based glycan analysis workflow for urinary PSA analysis. The technology utilizes selective, high yield single domain antibody based PSA capture, followed by preconcentration and capillary electrophoresis coupled with laser-induced fluorescence detection, resulting in high resolution N-glycan profiles. Urinary PSA glycan profiles were compared to a commercially available PSA standard revealing differences in their α2,3- and α2,6-sialylated isomers, proving the excellent selectivity of the suggested workflow. This is important as sialylation classification plays an important role in the differentiation between indolent, significant and aggressive forms of prostate cancer.

Separation based characterization methods for the N-glycosylation analysis of prostate-specific antigen.

Prostate cancer has the highest malignancy rate diagnosed in men worldwide. Albeit, the gold standard serum prostate-specific antigen (PSA) assays reduced the mortality rate of the disease, the number of false positive diagnoses steeply increased. Therefore, there is an urgent need for complementary biomarkers to enhance the specificity and selectivity of current diagnostic methods. Information about PSA glycosylation can help to fulfill this gap as alterations of its carbohydrate moieties due to cancerous transformation may represent additional markers to distinguish malignant from benign tumors. However, development of suitable methods and instrumentations to investigate the N-glycosylation profile of PSA represents a challenge. In this paper, we critically review the current bioanalytical trends and strategies in the field of PSA glycobiomarker research focusing on separation based characterization methods.

On-line enrichment of N-glycans by immobilized metal-affinity monolith for capillary electrophoresis analysis.

A novel N-glycan enrichment strategy is presented using unexpected but strong interactions between the sulfonate groups brought by the fluorescent dye of glycans and the Zr4+ modified poly(ethylene glycol methacrylate phosphate (EGMP)-co-acrylamide (AM)-co-bis-acrylamide (BAA)) monolith. The poly (EGMP-co-AM-co-BAA) monolith was synthesized via ultraviolet (UV) irradiation and then functionalized with Zr4+. The obtained monolith was characterized with scanning electron microscopy and mercury intrusion porosimetry. Large through-pores and a continuous skeleton with high permeability were observed. The N-glycans were labeled with the 1-aminopyrene-3, 6, 8-trisulfonic acid (APTS) and enriched by the Zr4+ modified monolith through IMAC interaction. This enrichment step was then coupled off-line to capillary electrophoresis (CE) separation with laser induced fluorescence (LIF) detection. Successful preconcentration of the APTS labeled maltooligosaccharide ladder was achieved under optimized conditions. Enrichment factors obtained for the maltooligosaccharides ranged from 9 to 24 with RSDs from 2.0% to 9.2% (n = 3). Moreover, very good repeatabilities (<6.7%) were obtained for glucose oligomers (4-15 glucose units) corresponding to sizes expected for N-glycans, demonstrating the great potential of this Zr4+ modified monolith to enrich APTS labeled glycans from N-glycoproteins. The proposed method was then successfully applied for the enrichment of N-glycans released from Ribonuclease B, in which case all five expected oligomannose glycans (Man 5 to Man 9) were successfully enriched. Thanks to the advantage of the method to enrich selectively APTS-glycans compared to the commercial SPE columns composed of HILIC or PGC materials, the first proof of concept of on-line enrichment coupled to CE-LIF separation was demonstrated for maltooligosaccharides as well.

Glycoprotein biomarkers and analysis in chronic obstructive pulmonary disease and lung cancer with special focus on serum immunoglobulin G.

Chronic obstructive pulmonary disease (COPD) and lung cancer are two major diseases of the lung with high rate of mortality, mostly among tobacco smokers. The glycosylation patterns of various plasma proteins show significant changes in COPD and subsequent hypoxia, inflammation and lung cancer, providing promising opportunities for screening aberrant glycan structures contribute to early detection of both diseases. Glycoproteins associated with COPD and lung cancer consist of highly sialylated N-glycans, which play an important role in inflammation whereby hypoxia leads to accumulation of sialyl Lewis A and X glycans. Although COPD is an inflammatory disease, it is an independent risk factor for lung cancer. Marked decrease in galactosylation of plasma immunoglobulin G (IgG) together with increased presence of sialic acids and more complex highly branched N-glycan structures are characteristic for COPD and lung cancer. Numerous glycan biomarkers have been discovered, and analysis of glycovariants associated with COPD and lung cancer has been carried out. In this paper we review fundamental glycosylation changes in COPD and lung cancer glycoproteins, focusing on IgG to provide an opportunity to distinguish between the two diseases at the glycoprotein level with diagnostic value.

Evaporative fluorophore labeling of carbohydrates via reductive amination.

As analytical glycomics became to prominence, newer and more efficient sample preparation methods are being developed. Albeit, numerous reductive amination based carbohydrate labeling protocols have been reported in the literature, the preferred way to conduct the reaction is in closed vials. Here we report on a novel evaporative labeling protocol with the great advantage of continuously concentrating the reagents during the tagging reaction, therefore accommodating to reach the optimal reagent concentrations for a wide range of glycan structures in a complex mixture. The optimized conditions of the evaporative labeling process minimized sialylation loss, otherwise representing a major issue in reductive amination based carbohydrate tagging. In addition, complete and uniform dispersion of dry samples was obtained by supplementing the low volume labeling mixtures (several microliters) with the addition of extra solvent (e.g., THF). Evaporative labeling is an automation-friendly glycan labeling method, suitable for standard open 96 well plate format operation.