Accurate Determination of Human Serum Transferrin Isoforms: Exploring Metal-Specific Isotope Dilution Analysis as a Quantitative Proteomic Tool

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• 作者：M. Estela del Castillo Busto ; Maria Montes-Bayó ; n ; Alfredo Sanz-Medel
• 刊名：Analytical Chemistry
• 出版年：2006
• 出版时间：December 15, 2006
• 年：2006
• 卷：78
• 期：24
• 页码：8218 - 8226
• 全文大小：247K
• 年卷期：v.78,no.24(December 15, 2006)
• ISSN：1520-6882

文摘

Carbohydrate-deficient transferrin (CDT) measurementsare considered a reliable marker for chronic alcoholconsumption, and its use is becoming extensive in forensic medicine. However, CDT is not a single molecularentity but refers to a group of sialic acid-deficient transferrin isoforms from mono- to trisialotransferrin. Thus,the development of methods to analyze accurately andprecisely individual transferrin isoforms in biologicalfluids such as serum is of increasing importance. Thepresent work illustrates the use of ICPMS isotope dilutionanalysis for the quantification of transferrin isoforms oncesaturated with iron and separated by anion exchangechromatography (Mono Q 5/50) using a mobile phaseconsisting of a gradient of ammonium acetate (0-250mM) in 25 mM Tris-acetic acid (pH 6.5). Species-specificand species-unspecific spikes have been explored. In thefirst part of the study, the use of postcolumn addition ofa solution of 200 ng mL^-1 isotopically enriched iron (⁵⁷Fe, 95%) in 25 mM sodium citrate/citric acid (pH 4)permitted the quantification of individual sialoforms oftransferrin (from S₂ to S₅) in human serum samples ofhealthy individuals as well as alcoholic patients. Second,the species-specific spike method was performed bysynthesizing an isotopically enriched standard of saturatedtransferrin (saturated with ⁵⁷Fe). The characterization ofthe spike was performed by postcolumn reverse isotopedilution analysis (this is, by postcolumn addition of asolution of 200 ng mL^-1 natural iron in sodium citrate/citric acid of pH 4). Also, the stability of the transferrinspike was tested during one week with negligible speciestransformation. Finally, the enriched transferrin was usedto quantify the individual isoforms in the same serumsamples obtaining results comparative to those of postcolumn isotope dilution and to those previously publishedin the literature, demonstrating the suitability of bothstrategies for quantitative transferrin isoform determination in real samples.