Mass spectrometric characteristics and kinetics of iron release in visceral mass of Saccostrea cucullata

Abstract

Ferritins with electrophoretic homogeneity were prepared from the visceral mass of Saccostrea cucullata in batch. The native PAGE approach showed similar electrophoretic mobility among pig pancreatic ferritin, liver ferritin of Dasyatis akajei, and visceral mass ferritin of Saccostrea cucullata. SDS-PAGE indicated that the Saccostrea cucullata visceral ferritin (SCVF) consisted of a single subunit type and had a molecular weight (MW) of approximately 20 kDa, suggesting that the protein shell in SCVF was composed of a single subunit. In addition, peptide mass fingerprinting and transmission electron microscopy were used to identify SCVF further, and to observe its molecular structure. We found that the molecular structure in SCVF was similar to that of most mammalian ferritins, which are composed of a protein shell and an iron core. The results of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry under the assistance of an acidic matrix, sinapic acid, also showed that SCVF was composed of a single subunit type and its subunit MW was calculated to be 19871.042 Da in the absence of heme. Kinetics analysis revealed that the complete process of iron release fitted the law of a first-order reaction, which is similar to that of most ferritins in mammals. Similar to bacterial ferritin, studies indicated that the shell consisted of a single subunit type and showed similar kinetics of iron release, suggesting that this subunit plays two important roles in iron release and storage, and that it shows different stability and intensity of interaction in carrying out its physiological functions in SCVF.

Determination of Iron Content and Dispersity of Intact Ferritin by Superconducting Tunnel Junction Cryodetection Mass Spectrometry

“…40 Other groups have studied characteristics of the ferritin subunits using mass spectrometric proteomic techniques. [41][42][43] The lack of MS publications on intact ferritin is likely due to the fact that traditional MS techniques for HoloFt could not be utilized because of the high molecular weight and broad size distribution of the assembly. Here we present heavy ion mass 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 7 spectrometry (HIMS) work above 100 kTh utilizing matrix-assisted laser desorption ionization (MALDI) time-of-flight (TOF) MS which incorporates a superconducting tunnel junction (STJ) cryodetector.…”

“…In 2006, de Val et al measured the mass of horse ApoFt, made recombinately consisting entirely of the L subunit, to be 491.5 kDa, also by ESI-MS . Other researchers have investigated modifications made to ApoFt/HoloFt in vitro . − Hoppler et al. have developed a quantitative method for measuring ferritin-bound iron in plant species utilizing isotope dilution MS .…”

“…have developed a quantitative method for measuring ferritin-bound iron in plant species utilizing isotope dilution MS . Other groups have studied characteristics of the ferritin subunits using mass spectrometric proteomic techniques. − The lack of MS publications on intact ferritin is likely due to the fact that traditional MS techniques for HoloFt could not be utilized because of the high molecular weight and broad size distribution of the assembly. Here we present heavy ion mass spectrometry (HIMS) work above 100 kTh utilizing matrix-assisted laser desorption ionization (MALDI) time-of-flight (TOF) MS which incorporates a superconducting tunnel junction (STJ) cryodetector.…”