Chip-based Reversed-phase Liquid Chromatography−Mass Spectrometry of Permethylated N-Linked Glycans: A Potential Methodology for Cancer-biomarker Discovery

Abstract

The study of protein glycosylation in biological fluids and tissues has substantial medical importance, as changes in glycan structures have now been associated with a number of diseases. Quantification of glycomic-profile changes is becoming increasingly important in the search for disease biomarkers. Here, we report a highly reproducible combination of a glycomic sample preparation/solid-phase derivatization of glycoprotein-derived N-linked glycans with their subsequent microchip-based separation and mass-spectrometric (MS) measurements. Following our previously-described reductive β-elimination for O-linked glycans with ammonia-borane complex to reduce N-linked structures, the N-linked alditol structures are effectively methylated in dimethylformamide medium to avoid artefacts in MS measurements. Reversed-phase microfluidic liquid chromatography (LC) of methylated N-linked oligosaccharide alditols resolved some closely related structures into regular retention increments, aiding in their structural assignments. Optimized LC gradients, together with nanospray MS, have been applied here in the quantitative measurements of N-linked glycans in blood serum, distinguishing breast cancer patients from control individuals.

Glycomic and Proteomic Profiling of Pancreatic Cyst Fluids Identifies Hyperfucosylated Lactosamines on the N-linked Glycans of Overexpressed Glycoproteins

“…Solid phase permethylation was performed using the spin column technique developed in our laboratory (18,19). The samples were first dissolved in 70 l of dimethylformamide, 5 l of water, and 25 l of iodomethane.…”

New Trends in the Study of Protein Glycosylation in Oncological Diseases

“…MS analýza může nebo nemusí používat derivatizaci vzorků, i když bylo mnohokrát prokázáno, že permetylace na pevné fázi [29] přináší jasné výhody co do zvýšené citlivosti a zahrnutí neutrálních i kyselých glykanových struktur v jednom analytickém měření. Různé detekční strategie pro LC a CE využívají mikrochemické navázání fl uoroforu ke glykanům, které jinak neobsahují jednoduše detekovatelné motivy ve svých molekulách.…”

Recent Developments in Liquid Chromatography and Capillary Electrophoresis for the Analysis of Glycoprotein Glycans

“…46 A combination of GCB and C18 SPE columns has been used for the purification of glycans for methylation analysis. 47 Borate can be used to form a diester complex with vicinal diols of saccharides at a pH value greater than 8. Therefore, an anion exchange resin of borate form is the most straightforward method to trap saccharides.…”