Details

Autor(en) / Beteiligte

• Farsang, Robert
• Jarvas, Gabor
• Guttman, Andras

Titel

Purification free N-glycan analysis by capillary zone electrophoresis: Hunt for the lost glycans

Ist Teil von

• Journal of pharmaceutical and biomedical analysis, 2024-01, Vol.238, p.115812-115812, Article 115812

Ort / Verlag

Elsevier B.V

Erscheinungsjahr

2024

Quelle

Access via ScienceDirect (Elsevier)

Beschreibungen/Notizen

• Capillary gel electrophoresis is a widely used method for rapid separation of fluorophore labeled carbohydrates. Even though, many publications conferred about this popular technique, no report yet investigated the possible sample losses during the purification process of the fluorophore labeling reaction mixture. In the present work, normal polarity capillary zone electrophoresis separation mode was applied to take advantage of the opposite migration directions of the electroosmotic flow and the negatively charged sample components using Tris-hexanoic acid running buffer at basic pH. For purification free oligosaccharide analysis, the separation parameters were designed in such a way that the triple charged labeling reagent of aminopyrenetrisulfonate (APTS) could not enter the separation capillary in contrary to the labeled sample components of interest, therefore, the APTS did not have to be removed before analysis. The method was used to show electrophoretic profile differences possibly caused by the cleanup process that was immediately apparent by comparing the electropherograms of the purified and non-purified APTS labeled maltooligosaccharides. Furthermore, qualitative and quantitative N-glycosylation profile alterations were revealed during CZE separation of the fluorophore labeling reaction mixtures before and after purification along with the analysis of the consecutively used washing solutions for the well characterized standard glycoproteins of IgG, ribonuclease B and fetuin. [Display omitted] •The CZE profile of APTS-maltooligosaccharides differed after purification.•At basic pH the sample components entered the capillary but the APTS not.•Separation parameters were established using the maltooligosaccharides.•The method was applied to analyze the washing fluids during the purification steps.•N-glycans pools from glycoproteins were analyzed before and after purification.