Characterizing the iron loading pattern of ferritin using high-mass matrix-assisted laser desorption ionization mass...

Ferritin is an iron storage protein assembly, usually formed by a 24-subunit protein shell and an iron core. The ferritin shell has been well studied using various structural biology tools such as X-ray diffraction and cryo-electron microscopy, whereas the iron status of ferritin is less studied and no well-established method exists for characterizing the distribution of the iron loading of ferritin. Recent advances in mass spectrometry (MS) have expanded the observable m/z range, making the measurement of ferritin possible with MS. In this study, matrix-assisted laser desorption ionization (MALDI)-MS was employed to quantify the iron content of ferritin.

The iron content of ferritin was quantified using a MALDI-MS system coupled with a commercially available ion conversions dynode high-mass detector. IgG1 antibody and its aggregates were used as external mass calibrants. The stability of HoloFt and ApoFt was also assessed in this study under different conditions, including various buffer pH, crosslinking agents and MALDI laser intensities.

The differences in peak width of HoloFt, ApoFt and IgG1 indicate the existence of mineral adducts in both HoloFt and ApoFt, and the mineral loading is heterogeneous among the HoloFt and ApoFt population. An average of 2773 ± 1584 iron atoms were determined for a commercial HoloFt sample. The iron core inside the ferritin complex is shown to stabilize and maintain the intact globular complex structure of ferritin.

This work introduces a MALDI-MS-based workflow for characterizing the ferritin iron loading pattern, which is meaningful for clinical analysis of iron deficiency/overload. In addition, the stability of ferritin is examined under various conditions, providing a guideline for further method development related to ferritin complex.