Time-of-flight mass spectrometry analysis of the ferroportin-hepcidin binding domain complex for accurate mass confirmation of bioactive hepcidin 25.
Time-of-flight mass spectrometry analysis of the ferroportin-hepcidin binding domain complex for accurate mass confirmation of bioactive hepcidin 25.
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对铁转运蛋白-铁调素结合域复合物进行飞行时间质谱分析，以准确质量确认生物活性铁调素 25。
DOI：
10.1016/j.cca.2009.11.031
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发表时间：
2010
期刊：
影响因子：
--
通讯作者：
Rockwood,AlanL
中科院分区：
文献类型：
--
作者：
Crockett,DavidK;Kushnir,MarkM;Phillips,JohnD;Rockwood,AlanL
In 2008, De Domenico et al. reported details of a binding assay to measure hepcidin bioactivity in serum [16]. By design, this functional binding assay measures only bioactive (defined here as ferroportin interacting) hepcidin 25 present in human serum. It was noted that the binding assay does not detect hepcidin in urine, which may suggest some difference in mass or structure of hepcidin 25 as it passes through the kidney and is eliminated from the body. Although several methods have been reported to measure hepcidin, important questions remain such as; what form of hepcidin is bioactive? What are the specific differences between serum and urine hepcidin? What are the primary causes of the loss of biological activity? To date, accurate mass confirmation of the ferroportin bound form of hepcidin has not been reported.In brief, ferroportin hepcidin binding domain (HBD) peptides (RRFDCITTGYAYTQGLSGSILSRR—MW 2720.38) were immobilized covalently to agarose beads using the AminoLink Plus Immobilization Kit (Pierce, Rockford IL). An aliquot (100 μl) of human serum or urine was added to HBD-conjugated beads. The samples (n= 4) were incubated at 37 C for 12–18 h, the beads were washed, then bound and unbound fractions collected. The use of human serum and urine samples was approved by the University of Utah Institutional Review Board. After the affinity capture, bound and unbound fractions were prepared for analyses as reported by Merrell et al.[17]. Briefly, 100 μl of each sample was mixed with 200 μl acetonitrile for 30 s, incubated at room temperature for 30 min, and centrifuged for 10 min at 15,000× g. Supernatant was removed, lyophilized and reconstituted in 10 μl water containing 0.1% formic acid. All samples remained above 25 C until analysis to avoid premature dissociation of the HBD/hepcidin complex [16].
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