New insights in carbohydrate-deficient transferrin analysis with capillary electrophoresis-mass spectrometry.
Capillary zone electrophoresis (CZE) with UV detection has been widely used for the determination of carbohydrate-deficient transferrin (CDT), an indirect marker of the chronic alcohol consumption (≥60-80g/day). A commercially available method (CEofix™ CDT kit), containing a bilayer anionic coating, allows for the analysis of CDT with a high resolution between transferrin (Tf) glycoforms with reduced protein adsorption onto the capillary wall. Although widely used in routine analysis, this procedure presents some limitations in terms of selectivity and sensitivity which may be overcome with mass spectrometry (MS). However, the available method is not MS-compatible due to the non-volatile coating as well as the phosphate and borate buffers present in the background electrolyte (BGE). This study firstly consisted in developing MS-compatible separation conditions, i.e., coating and BGE compositions. Numerous cationic, neutral, and anionic coatings were evaluated in combination with BGEs covering a broad range of pH values. A bilayer coating composed of a cationic layer of 10% polybrene (m/v) and an anionic layer of 10% dextran sulfate (m/v) combined with a BGE composed of 20mM ammonium acetate at pH 8.5 provided the best results in terms of glycoforms' resolution, efficiency, adsorption reduction, migration times' repeatability, and coating stability. The method was then transferred to CZE-MS after investigations of the electrospray ionization (ESI) source, equipped with a sheath-flow interface, and the time-of-flight (TOF/MS) parameters. A successful MS detection of tetrasialo-Tf was obtained during infusion, while the experiments highlighted the challenges and issues encountered with intact glycoprotein analysis by CZE-ESI-MS.
School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Bd d'Yvoy 20, 1211 Geneva 4, Switzerland; Swiss Centre for Applied Human Toxicology, University of Geneva, CMU, Rue Michel-Servet 1, 1211 Geneva 4, Switzerland.
Isabelle
I
Kohler
Swiss Centre for Applied Human Toxicology, University of Geneva, CMU, Rue Michel-Servet 1, 1211 Geneva 4, Switzerland; University Center of Legal Medicine (CURML), Lausanne-Geneva, Rue du Bugnon 21, 1011 Lausanne, Switzerland.
Marc
M
Augsburger
School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Bd d'Yvoy 20, 1211 Geneva 4, Switzerland; Swiss Centre for Applied Human Toxicology, University of Geneva, CMU, Rue Michel-Servet 1, 1211 Geneva 4, Switzerland.
Serge
S
Rudaz
School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Bd d'Yvoy 20, 1211 Geneva 4, Switzerland; Swiss Centre for Applied Human Toxicology, University of Geneva, CMU, Rue Michel-Servet 1, 1211 Geneva 4, Switzerland. Electronic address: julie.schappler@unige.ch.
Julie
J
Schappler
D000437:Alcoholism / Q000175:diagnosis; D000838:Anions; D015415:Biomarkers / Q000032:analysis; D002412:Cations; D019075:Electrophoresis, Capillary / Q000379:methods*; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D013499:Surface Properties; D014168:Transferrin / Q000031:analogs & derivatives* / Q000032:analysis
D000838:Anions; D015415:Biomarkers; D002412:Cations; D014168:Transferrin; C056759:carbohydrate-deficient transferrin
Capillary coating; Capillary electrophoresis; Carbohydrate-deficient transferrin; Chronic alcohol consumption; Mass spectrometry