Subunit Characteristics of Pig Pancreas Ferritin Revealed by MALDI-TOF MS and RP-HPLC

Abstract

Pig pancreas ferritin(PPF) was purified by ultra-centrifugation, ion-exchange chromatography, and native gradient polyacrylamide gel electrophoresis(PAGE_NG). Sodium dodecyl sulfate(SDS)-PAGE indicates that PPF consists of two subunit types, namely, H(21000) and L(19000) subunits, and its core shows an average element composition of 1698 Fe³⁺ and 179 phosphate molecules within the hollow shell, giving a 9.5:1 ratio of Fe³⁺ to phosphate. An off line approach combining reversed-phase high-performance liquid chromatography(RP-HPLC) with matrix-assisted laser desorption ionization time of flight mass spectrometry(MALDI-TOF MS) made the decomposition of PPF shell into H and L subunits for the analysis of mass spectrometry(MS), giving molecular weights of both H(21014.4) and L(18319.9) subunits. Both subunit types were further identified by an approach combining peptide mass fingerprint(PMF) with database search. A ratio of 1H to 2L subunits in PPF was determined by SDS-PAGE, RP-HPLC, and MALDI-TOF MS, respectively. It is well known that the non-covalent interaction of L-L or H-L subunits is stronger than that of H-H subunits in PPF, which may be further used to explain the unclear physiological function between H and L subunits in PPF.