Development of a high‐throughput glycoanalysis method for the characterization of oligosaccharides in human milk utilizing multiplexed capillary gel electrophoresis with laser‐induced fluorescence detection

Abstract

During the last decade, enormous progress regarding knowledge about composition and properties of human milk (HM) has been made. Besides nutrition, the three macro-nutrients: proteins, lipids, and carbohydrates combine a large variety of properties and functions. Especially, complex oligosaccharides emerge as important dietary factors during early life with multiple functions. The characterization of these HM oligosaccharides (HMOS) within the total carbohydrate fraction is prerequisite to understand the relationship between milk composition and biological effects. Therefore, extended studies of large donor cohorts and thus, new high-throughput glycoanalytical methods are needed. The developed method comprises sample preparation, as well as analysis of HMOS by multiplexed CGE with LIF detection (xCGE-LIF). Via a respective database the generated "fingerprints" (normalized electropherograms) could be used for structural elucidation of HMOS. The method was tested on HM samples from five different donors, partly sampled as a series of lactation time points. HMOS could be easily identified and quantified. Consequently, secretor and Lewis status of the donors could be determined, milk typing could be performed and quantitative changes could be monitored along lactation time course. The developed xCGE-LIF based "real" high-throughput HMOS analysis method enables qualitative and quantitative high-performance profiling of the total carbohydrate fraction composition of large sets of samples.

Prognostic Value of Plasma and Urine Glycosaminoglycan Scores in Clear Cell Renal Cell Carcinoma

“…All samples were stored at −80°C until they were shipped for analysis in dry ice. GAG measurements were conducted using capillary electrophoresis with laser-induced fluorescence, as previously described (16, 17). Based on these measurements, the plasma and urine biomarkers were scored according to formula derived previously (14) and here reported: $\text{Plasma score}=\frac{[6{\text{s CS]}+\text{CS}}_{\text{tot}}}{\frac{3}{10}\frac{\text{[4s CS]}}{[6\text{s CS]}}+[\text{Ns HS]}}$ $\text{Urine score}=\frac{\left[\text{Ns6s HS}\right]+60·\text{Charge HS}}{\left[4\text{s CS}\right]},$ where [6s CS] represents the fraction of the 6-sulfated chondroitin sulfate, [4s CS] represents the fraction of the 4-sulfated chondroitin sulfate, [Ns6s HS] represents the fraction of the N-sulfated 6-sulfated heparan sulfate, [Ns HS] represents the fraction of the N-sulfated heparan sulfate, CS tot is the total concentration of CS (in micrograms per milliliter), and Charge HS is the total fraction of sulfated disaccharides of HS.…”

Analysis of carbohydrates and glycoconjugates by matrix‐assisted laser desorption/ionization mass spectrometry: An update for 2013–2014

“…A method for high‐throughput analysis of oligosaccharides in human milk has been reported by Kottler et al () Briefly, the milk was pasteurized, skimmed, and deproteinized to give a carbohydrate fraction which was purified by gel‐permeation chromatography and reversed‐phase HPLC. Analysis was by MALDI‐MS/MS, LC‐MS, and gel electrophoresis with laser‐induced fluorescence.…”

Capillary electrophoresis separation of human milk neutral and acidic oligosaccharides derivatized with 2‐aminoacridone

“…CE with LIF coupled to MS was found to be effective for the separation and characterization of 9‐aminopyrene‐1,4,6‐trisulfonate derivatized HMOs . Very recently, a high‐throughput method for the characterization of HMOs using multiplexed CGE‐LIF has been developed . However, the introduction of a fluorophore in the HMO molecule add a group for fluorescence or UV detection but also useful to simultaneously improve separation.…”