A general approach for the purification and quantitative glycomic analysis of human plasma
| Autor: | William S. Hancock, Samnang Tep, Marina Hincapie |
|---|---|
| Rok vydání: | 2012 |
| Předmět: |
Chromatography
Glycan Glycosylation biology Blood Proteins Biochemistry Reductive amination Mass Spectrometry Analytical Chemistry carbohydrates (lipids) Glycomics chemistry.chemical_compound Matrix-assisted laser desorption/ionization chemistry Polysaccharides Anthranilic acid biology.protein Humans Derivatization Quantitative analysis (chemistry) Glycoproteins |
| Zdroj: | Analytical and Bioanalytical Chemistry. 402:2687-2700 |
| ISSN: | 1618-2650 1618-2642 |
| Popis: | The development of a general method for the purification and quantitative glycomic analysis of human plasma samples to characterize global glycosylation changes shall be presented. The method involves multiple steps, including the depletion of plasma via multi-affinity chromatography to remove high abundant proteins, the enrichment of the lower abundant glycoproteins via multi-lectin affinity chromatography, the isotopic derivatization of released glycans, and quantitative analysis by MALDI-TOF MS. Isotopic derivatization of glycans is accomplished using the well-established chemistry of reductive amination to derivatize glycans with either a light analog ((12)C anthranilic acid) or a heavy analog ((13)C(7) anthranilic acid), which allows for the direct comparison of the alternately labeled glycans by MALDI-TOF MS. The method displays a tenfold linear dynamic range for both neutral and sialylated glycans with sub-picomolar sensitivity. Additionally, by using anthranilic acid, a very sensitive fluorophore, as the derivatization reagent, the glycans can be analyzed by chromatography with fluorescence detection. The utility of this methodology is highlighted by the many diseases and disorders that are known to either show or be the result of changes in glycosylation. A method that provides a generic approach for sample preparation and quantitative data will help to further advance the field of glycomics. |
| Databáze: | OpenAIRE |
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