Analysis of human transferrin glycopeptides by capillary electrophoresis and capillary liquid chromatography-mass spectrometry. Application to diagnosis of alcohol dependence

• CE-TOF-MS and μLC-TOF-MS methods separate and detect Tf glycopeptide glycoforms.
• The surfactant used to enhance the enzymatic digestion interferes in CE analyses.
• Tf alteration in alcoholism is mainly due to entire loss of the carbohydrate chains.
• Ethanol intake has no effect in the activity of sialyltransferase and sialidase.
• This method could be used to elucidate Tf glycosylation in different subtypes of CDGs.

In this study, capillary electrophoresis and capillary liquid chromatography coupled to mass spectrometry (CE-TOF-MS and μLC-TOF-MS) were used to detect and characterise human transferrin (Tf) glycopeptide glycoforms obtained by tryptic digestion. After selecting μLC-TOF-MS because of improved performance in analysis of N413 and N611 glycopeptide glycoforms, the proposed methodology was applied to serum samples. Two immunoaffinity columns were employed to isolate Tf from serum samples. Both columns were activated with the same anti-Tf antibody but using two different bonding chemistries. After immunoaffinity purification and digestion, serum samples from a teetotal individual (as control) and from individuals with low and high alcohol dependence were analysed by μLC-TOF-MS. Relative abundance of each glycoform was useful to estimate the degree of alcohol dependence of each individual. Finally, the established methodology was used to analyse serum samples from specific individuals with an unknown degree of alcohol dependence.

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