Comparison of different techniques for the analysis of metallothionein isoforms by capillary electrophoresis |
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| Institution: | 1. USDA, Agricultural Research Service, Growth Biology Laboratory, Beltsville, MD 20705-2350, USA;2. Division of Biochemical Sciences, Rowett Research Institute, Bucksburn, Aberdeen, AB2 9SB, UK;1. St. Petersburg State University, Institute of Chemistry, Universitetskii pr. 26, 198504, St. Petersburg, Russia;2. Institute of Macromolecular Compounds, Russian Academy of Sciences, Bolshoy pr. 31, 199004, St. Petersburg, Russia;1. Institute for Environmental and Climate Research, Jinan University, Guangzhou 511443, China;2. The National Center for Chronic and Noncommunicable Disease Control and Prevention, Beijing 100050, China;3. Department of Epidemiology and Biostatistics, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine, Peking Union Medical College, Beijing 100005, China;4. Department of Health, Ethics & Society, CAPHRI School of Public Health and Primary Care, Faculty of Health, Medicine and Life Sciences, Maastricht University, The Netherlands;5. Centre for Infectious Diseases, Epidemiology and Surveillance, National Institute for Public Health and the Environment, Bilthoven, The Netherlands;6. State Key Laboratory of Infectious Disease Prevention and Control, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China |
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| Abstract: | We have investigated free-solution capillary electrophoresis (FSCE) and micellar electrokinetic capillary chromatography (MECC) separations of metallothionein (MT) isoforms conducted in uncoated and surface-modified fused-silica capillaries. At alkaline pH, FSCE rapidly resolves isoforms belonging to the MT-1 and MT-2 charge classes. At acidic pH, additional resolution of MT isoforms is achieved. The use of high-ionic-strength (0.5 M) phosphate buffers can result in high peak efficiencies and increased resolution for some MT isoforms. Interior capillary surface coatings such as polyamine and linear polyacrylamide polymers permit separation of MT isoforms with enhanced resolution through their effects on electroosmotic flow (EOF) and protein-wall interactions. Improvements in MT isoform resolution can also be achieved by MECC using 100 mM borate buffer pH 8.4 containing 75 mM SDS. Deproteinization of tissue cytosol samples with acetonitrile (60–80%) or perchloric acid (7%) produces extracts that can be subjected to direct analysis of MT by FSCE or MECC. We conclude that optimal separation of MT isoforms by capillary electrophoresis (CE) can be achieved with the appropriate combination of different capillaries, buffers and sample preparation techniques. |
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