Full-length L- and H-subunits and a truncated L-subunit are detected in serum ferritin. (A) The main ferritin peak from Figure 1B was concentrated and separated on sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). Alternatively, serum ferritin was immunoprecipitated and separated on SDS-PAGE. Relevant protein bands were analyzed by LC-MS/MS mass spectrometry. Black-labeled amino acid sequences were detected by MS, showing the presence of L-, H- and a truncated L-subunit. L-subunit contains 1 potential site for N-glycosylation at the N-terminus of the protein (underlined) that faces the exterior surface of the assembled protein (determined by comparison to the human 24 subunit assembled structure; http://www.ncbi.nlm.nih.gov/Structure/CN3D/cn3d.shtml). (B) The structure of the L-subunit sequences was predicted using the Swiss Model Web site (http://www.ebi.ac.uk/swissprot). The truncated L- ferritin lacks the entire fifth short helix at the C-terminal end of the protein. (C) Top panel: Coomassie staining of immunoprecipitated liver, heart, and serum ferritin from iron-overloaded mice detects L-subunit very clearly while H-subunit in serum is hardly detectable. A 17-kD band (marked) S-subunit is prominent in serum (1 representative experiment is shown of 5). Middle and bottom panels: Immunoprecipitation of 5 μg of liver, heart, and serum ferritin with an anti–mouse liver ferritin antibody followed by Western blot with the same anti–liver antibody (middle panel) or with an anti–H-subunit antibody (bottom panel) shows that serum ferritin contains many fewer H-subunits than liver and heart ferritin (1 representative experiment is shown of 3 for L ferritin Western and of 6 for H-ferritin Western). It is important to note that in contrast to human ferritin, mouse L-ferritin subunits migrate at around 21 kD and H-ferritin subunits migrate slightly faster.