N-Glycan profile analysis of transferrin using a microfluidic compact disc and MALDI-MS

Abstract

It has been known for a long time that diseases can be associated with changes to the glycosylation of specific proteins. This has been shown for cancer, immunological disorders, and neurodegenerative diseases. The possibility of using the glycosylation patterns of proteins as biomarkers for disease would be a great asset for clinical research or diagnosis. There is at present a lack of rapid, automated, and cost-efficient analytical techniques for the determination of the glycosylation of specific serum proteins. We have developed a method for determining the glycosylation pattern of proteins based on the affinity capture of a specific serum protein, the enzymatic release of the N-linked glycans, and the analysis of the glycan pattern using MALDI-MS. All sample preparation is performed in a disposable centrifugal microfluidic disc. The sample preparation is miniaturized, requiring only 1 μL of sample per determination, and automated with the possibility of processing 54 samples in parallel in 3.5 h. We have developed a method for the glycosylation pattern analysis of transferrin. The method has been tested on serum samples from chronic alcohol abusers and a control group. Also, a SIMCA model was created and evaluated to discriminate between the two groups.

Diagnostic Potential of Transferrin Glycoforms—A Lectin‐Based Protein Microarray Approach

“…ConA is specific for the high‐mannose (Man)‐type structures and hTf is known to possess mostly branched mannosidic structures . Interactions of the moderate intensity were detected between hTf and WGA, specific for N‐acetylglucosamine structures (GlcNAcβ1‐4GlcNAcβ1‐4GlcNAc) and to a lesser extent for Sia . Interactions between hTf and AAL or PHA‐E were relatively weak, as their signal intensities ranged from 500 to 300 a.u.…”

“…[37] Interactions of the moderate intensity were detected between hTf and WGA, specific for N-acetylglucosamine structures (GlcNAcβ1-4GlcNAcβ1-4GlcNAc) and to a lesser extent for Sia. [25,37,39] Interactions between hTf and AAL or PHA-E were relatively weak, as their signal intensities ranged from 500 to 300 a.u. respectively.…”

“…AAL is specific for the fucosylated (Fuc) structures, especially for the core α1,6-fucosylated oligosaccharides and hTf is known to have them. [25] PHA-E interacts with Gal and GlcNAc and can serve for the detection of bi-antennary complex type N-glycans with terminal Gal and/or bisecting GlcNAc. Both types of saccharides are present in hTf glycans.…”

“…Glycosylation of hTf was studied by HPLC, HILIC, MS, capillary electrophoresis, and affinity‐based techniques . MS is considered as the most sensitive method to study glycan structures, but it is time‐consuming and expensive approach, involving separation of glycans from the protein part of the molecule prior to MS analysis.…”

“…[19,20] Glycosylation of hTf was studied by HPLC, HILIC, MS, capillary electrophoresis, and affinity-based techniques. [21][22][23][24][25] MS is considered as the most sensitive method to study glycan structures, but it is time-consuming and expensive approach, involving separation of glycans from the protein part of the molecule prior to MS analysis. All mentioned techniques are focused on the total protein glycoprofile, involving enzymatically or chemically released glycans from proteins.…”