Identification of α₂ macroglobulin as a hepcidin-binding protein. (A) A hepcidin-binding protein in complete human plasma or fractionated plasma comigrates with a complex of purified α₂-macroglobulin (α₂-M) and ¹²⁵I-hepcidin. Lane 1: fractions 15 through 17 from the plasma fractionation experiment (see Figure 2C) were pooled and concentrated, separated by native gradient PAGE, and stained using Coomassie blue. Lanes 2 through 4 (phosphorimager scan of a different gel run in parallel to the Coomassie blue gel in lane 1): complete human plasma (lane 2), pooled fractions 15 through 17 (lane 3; from Figure 2C) and purified α₂-M (10 μg; lane 4) were incubated with ¹²⁵I-hepcidin (2.8 μM) for 1 hour at 37°C and separated by native PAGE and visualized using a phosphorimager. (B,C) Identification of the hepcidin-binding protein in blood plasma by mass spectrometry. MS/MS spectrum confirming the identity of α₂-M in the top band of panel A. The region of the gel corresponding to the top band that consists of ¹²⁵I-hepcidin bound to an unknown plasma protein from pooled fractions 15 through 17 (Figure 3A lane 3) was cut from the gel and digested with trypsin. The resulting tryptic peptides were separated on a C18 column connected online to the mass spectrometer. Each full scan experiment measuring the peptide masses was followed by 3 MS/MS fragmentation scans providing sequence information of the fragmented peptides. As a representative example of one peptide identification, the full MS scan (B) and fragmentation MS/MS spectrum of the peptide ⁴⁹³LSFYYLIMAK⁵⁰² (C) from α₂-M is shown. The MS spectrum exhibits intense signals at m/z 625.2 (circled) and 1248.3. These signals represent doubly- and singly-charged ions of the peptide, respectively. The near complete series of C-terminal y-ions in the MS/MS spectrum of doubly-charged ions clearly confirms the α₂-M peptide sequence ⁴⁹³LSFYYLIMAK⁵⁰² (panel C top line). The major peak in the full scan spectrum at m/z 923.2 corresponds to an additional coeluting peptide originating from α₂-M, as was confirmed by its MS/MS spectrum (not shown).