FIGURE 1.

Disulfide analysis of urinary hepcidin with partial reductive alkylation. A, after reduction at 37 °C for 8 min in 0.1% trifluoroacetic acid, pH 2, the reaction products were separated by reversed phase HPLC. The first broad peak (12–13 min) is a nonalkylated form of hepcidin. Both peaks 1 and 2 were major products, containing 2NEM-labeling groups. B, sequence analysis of peak 1 for determination of NEM-modified sites. Yield of PTH-NEM-Cys is indicated for all cysteines. NEM signal is detected for residues Cys⁵, Cys⁷, and Cys⁸. C, sequence results of peak 1 after CNBr treatment to remove the segment of the molecule that contains cysteines 1–6 (and hence lower the background) reveals that the second labeled species is Cys⁷. D, sequence analysis of peak 2 for determination of NEM-modified sites. NEM signal is detected for Cys², Cys⁴, and Cys⁵. The sequences in E represent disulfide connectivities determined from either B/C and are one-dimensional.