Hello,

There are a few different ways to approach your problem.

With 8 Cys present, hepcidin-25 should be electrochemically active. It may be that your sample is oxidized and no longer detectable. The easiest way to address this is to treat the samples (standards and biological0, first with a reducing reagent like dithiothreitol (DTT). Selvi shows how to do this with glutathione.

(Selvi, M. Bharath. "Biochemical Mechanisms of Homocysteine and its Role in RETINAL VASCULAR DISEASES." PhD diss., BITS Pilani, 2015.

See also: Sakhi, A.K., Russnes, K.M., Smeland, S., Blomhoff, R. and Gundersen, T.E., 2006. Simultaneous quantification of reduced and oxidized glutathione in plasma using a two-dimensional chromatographic system with parallel porous graphitized carbon columns coupled with fluorescence and coulometric electrochemical detection. Journal of Chromatography A, 1104(1-2), pp.179-189.

Zhang, M. and Pfeiffer, C.M., 2004. Comparing the ESA HPLC total homocysteine assay with electrochemical detection to the CDC in-house HPLC assay with fluorescence detection. Clinica chimica acta, 340(1-2), pp.195-200.}

Standards of glutathione or oxidized glutathione (10 µg/mL) are made in buffer. 50 µL of standard is mixed with 5 µL of 50 mM DTT at room temperature for 10 minutes. 10 µL of the mixture is injected into an HPLC (Buffer: 0.1% trifluoroacetic acid, 2% acetonitrile, 98% MilliQ water; Flow rate: 1 mL/min; Pressure: 400 bar; Temperature: 27.7 °C; ECD with glassy carbon electrode; Range: 1 µA; E cell: 0.90; Voltage: 1.16V; Column: ODS Hypersil C-18, 5 µm particle size, 150 X 4.6 mm).

Note that the moles of disulfide bonds per gram of oxidized GSSG is different than the moles of disulfide bonds (possible) in hepcidin-25 by ~1.75:1. So you might want to make the DTT solution a little stronger (~90 mM). You could also add more 50 mM DTT solution but that will dilute your sample.

Since you are open to other methods, I have attached a chapter on thiol-reactive fluorescent dyes that you can use to label Hepcidin-25. Again, remember the stoichiometry and be sure to first reduce any disulfide bonds in Hepcidin-25 (intra- or inter=molecular) and also use enough thiol-reactive dye to label all 8 Cys.

Best regards,

thanks, very much Joe, I will read these papers and try to come up with a plan.

It may be interesting to know that hepcidin-25 forms very strong copper(II) complexes under neutral and basic conditions. They most likely should be electrochemically active.

Article Investigations of the copper peptide hepcidin-25 by LC-MS/MS and NMR

Article Improved LC-MS/MS method for the quantification of hepcidin-...