RATIONALE 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. METHODS 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. RESULTS 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. CONCLUSIONS 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.

中文翻译：

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使用高质量基质辅助激光解吸电离质谱法表征铁蛋白的铁负载模式。

原理铁蛋白是铁存储蛋白装配体，通常由24个亚基蛋白壳和铁芯形成。已经使用各种结构生物学工具（例如X射线衍射和冷冻电子显微镜）对铁蛋白壳进行了充分的研究，而铁蛋白的铁状态研究较少，并且没有成熟的方法来表征铁蛋白的铁负载分布。质谱（MS）的最新进展扩大了可观察的m / z范围，从而使MS的铁蛋白测量成为可能。在这项研究中，采用基质辅助激光解吸电离（MALDI）-MS来定量铁蛋白的铁含量。方法使用MALDI-MS系统和可购得的离子转化倍增极高质量检测器，对铁蛋白的铁含量进行定量。IgG1抗体及其聚集体用作外部质量校准物。在这项研究中还评估了HoloFt和ApoFt在不同条件下的稳定性，包括不同的缓冲液pH值，交联剂和MALDI激光强度。结果HoloFt，ApoFt和IgG1峰宽的差异表明HoloFt和ApoFt中均存在矿物质加合物，并且HoloFt和ApoFt人群的矿物质负载是异质的。对于商业HoloFt样品，平均铁原子数为2773±1584。铁蛋白复合物内部的铁核显示稳定并维持铁蛋白的完整球状复合物结构。结论这项工作介绍了一种基于MALDI-MS的工作流程，用于表征铁蛋白铁的负载模式，这对铁缺乏/超负荷的临床分析具有重要意义。