A Targeted Mass Spectrometric Assay for Reliable Sensitive Hepcidin Quantification.
A Targeted Mass Spectrometric Assay for Reliable Sensitive Hepcidin Quantification.
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用于可靠、灵敏的铁调素定量的靶向质谱分析。
DOI：
10.1038/s41598-019-43756-9
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发表时间：
2019
影响因子：
4.6
通讯作者：
Ansong,Charles
中科院分区：
文献类型：
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作者：
Moghieb,Ahmed;Tesfay,Lia;Nie,Song;Gritsenko,Marina;Fillmore,ThomasL;Jacobs,JonM;Smith,RichardD;Torti,FrankM;Torti,SuzyV;Shi,Tujin;Ansong,Charles
铁调素是一种富含半胱氨酸的肽类激素，主要由肝脏分泌，在铁代谢调节中起核心作用。新出现的证据表明，铁代谢紊乱是包括癌症在内的各种疾病的危险因素。然而，由于完整铁调素的低片段化效率以及完整铁调素标准品的合成困难，应用当前基于质谱（MS）的铁调素测定进行精确定量仍然具有挑战性。为了解决这些问题，我们最近开发了一种可靠的灵敏的靶向MS测定法，用于从临床样品中定量铁调素，该测定法使用完全烷基化的而不是完整的铁调素作为内标。检测限和定量限分别确定为<0.5 ng/mL和1 ng/mL。将烷基化铁调素测定应用于70个临床血浆样品（42个非癌性和28个卵巢癌患者样品）使得能够可靠地检测来自血浆样品以及条件培养基的内源性铁调素。在非癌和癌症血浆样本中，铁调素浓度范围为0.0至95.6 ng/mL。有趣的是，与非癌患者相比，癌症患者的铁调素浓度显着更高（平均值：癌症为20.6 ng/ml;非癌患者为5.94 ng/ml）（p值< 0.001）。我们的研究结果代表了烷基化铁调素测定法在临床样品中的首次应用，并证明所开发的测定法具有比目前基于MS的铁调素测定法更好的灵敏度和定量准确度，而无需在合成完整铁调素标准品和准确测定其绝对量方面面临挑战。
Hepcidin, a cysteine-rich peptide hormone, secreted mainly by the liver, plays a central role in iron metabolism regulation. Emerging evidence suggests that disordered iron metabolism is a risk factor for various types of diseases including cancers. However, it remains challenging to apply current mass spectrometry (MS)-based hepcidin assays for precise quantification due to the low fragmentation efficiency of intact hepcidin as well as synthesis difficulties for the intact hepcidin standard. To address these issues we recently developed a reliable sensitive targeted MS assay for hepcidin quantification from clinical samples that uses fully alkylated rather than intact hepcidin as the internal standard. Limits of detection and quantification were determined to be <0.5 ng/mL and 1 ng/mL, respectively. Application of the alkylated hepcidin assay to 70 clinical plasma samples (42 non-cancerous and 28 ovarian cancer patient samples) enabled reliable detection of endogenous hepcidin from the plasma samples, as well as conditioned culture media. The hepcidin concentrations ranged from 0.0 to 95.6 ng/mL across non-cancerous and cancer plasma specimens. Interestingly, cancer patients were found to have significantly higher hepcidin concentrations compared to non-cancerous patients (mean: 20.6 ng/ml for cancer; 5.94 ng/ml for non-cancerous) (p value < 0.001). Our results represent the first application of the alkylated hepcidin assay to clinical samples and demonstrate that the developed assay has better sensitivity and quantification accuracy than current MS-based hepcidin assays without the challenges in synthesis of intact hepcidin standard and accurately determining its absolute amount.