新型锆离子固定化亲和磁纳米粒子的制备及在磷酸化肽高效捕获中的应用

引用本文: 赵艳艳, 张丽华, 张丽媛. 新型锆离子固定化亲和磁纳米粒子的制备及在磷酸化肽高效捕获中的应用[J]. 分析化学, 2020, 48(2): 233-239. doi: 10.19756/j.issn.0253-3820.191681 shu

Citation: ZHAO Yan-Yan, ZHANG Li-Hua, ZHANG Li-Yuan. Preparation of Novel Zirconium Ion immobilized Affinity Magnetic Nanoparticles and Application in Efficient Capture of Phosphopeptides[J]. Chinese Journal of Analytical Chemistry, 2020, 48(2): 233-239. doi: 10.19756/j.issn.0253-3820.191681 shu

新型锆离子固定化亲和磁纳米粒子的制备及在磷酸化肽高效捕获中的应用

1.
大连医科大学基础医学院, 大连 116044;
2.
中国科学院分离分析化学重点实验室, 中国科学院大连化学物理研究所, 大连 116023;
3.
大连医科大学药学院, 大连 116044
基金项目:
本文系国家自然科学基金项目（Nos.21974017，21505015）、中国科学院分离分析化学重点实验室开放基金项目、辽宁省教育厅科学研究一般项目（No.LZ2019063）、辽宁省自然科学基金项目（No.20170540286）和大连市支持高层次人才创新创业计划（No.2016RQ041）资助

摘要: 蛋白质磷酸化是最常见、最重要的翻译后修饰之一，在细胞信号传导、细胞凋亡等生物学过程中起关键的调控作用。但是，磷酸化蛋白/肽具有低丰度和负电荷的特点，在生物质谱常规的正离子模式下，易受到高丰度非磷酸化蛋白/肽的信号抑制，难以直接对其进行直接分析。因此，有必要在质谱分析前对生物样品中的磷酸化蛋白/肽进行选择性富集。本研究基于三磷酸腺苷（ATP）配基，制备了新型锆离子固定化亲和磁纳米粒子（Zr⁴⁺-ATP-MNP）。扫描电镜和元素分析的表征结果表明，Zr⁴⁺-ATP-MNP制备成功。采用所制备的Zr⁴⁺-ATP-MNP材料对磷酸化肽进行富集，以磷酸化蛋白β-酪蛋白（β-casein）的酶解液为选择性实验模式样品，采用质谱法可鉴定出9条磷酸化肽；以β-casein和非磷酸化蛋白牛血清白蛋白（BSA）酶解液（1∶100，n/n）混合物为干扰实验模式样品，采用质谱可鉴定出5条磷酸化肽。富集后，磷酸化肽的质谱信号强度显著提高，可有效去除非磷酸化肽。Zr⁴⁺-ATP-MNP在牛奶酶解产物中的应用进一步表明，其对复杂生物样品中的单磷酸化肽和多磷酸化肽具有广谱性的捕获潜力。

关键词:

固定化亲和
/ 磷酸化肽
/ 富集
/ 磁性

English

Preparation of Novel Zirconium Ion immobilized Affinity Magnetic Nanoparticles and Application in Efficient Capture of Phosphopeptides

1.
College of Basic Medical Sciences, Dalian Medical University, Dalian 116044, China;
2.
CAS Key Laboratory of Separation Sciences for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China;
3.
College of Pharmacy, Dalian Medical University, Dalian 116044, China
Received Date: 18 November 2019
Revised Date: 19 December 2019
Fund Project:
This work was supported by the National Natural Science Foundation of China (Nos. 21974017, 21505015), the Project of CAS Key Laboratory of Separation Sciences for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, the Program in Liaoning Province Education Department (No. LZ2019063), the Natural Science Foundation of Liaoning Province, China (No. 20170540286), and the Dalian High-level Talents Innovation Support Program (No. 20170540286).

Abstract: Protein phosphorylation is one of the most common and important post-translational modifications, which plays key regulatory roles in biological processes, such as cell signaling and apoptosis. However, due to the low abundance and negative charge of phosphorylated proteins, in the conventional positive ion mode of bio-mass spectrometry, phosphoproteins are susceptible to be suppressed by high-abundance non-phosphorylated proteins, making them difficult to be directly analyzed. Therefore, the enrichment of phosphorylated proteins/peptides prior to mass spectrometry is important in biological research. For the first time, in this study, a novel zirconium ion-immobilized affinity magnetic nanoparticle (Zr⁴⁺-ATP-MNP) was prepared using adenosine triphosphate (ATP) as the ligand. The ATP ligand in Zr⁴⁺-ATP-MNP contains three phosphate groups, which can simultaneously provide multiple zirconium ion chelating sites, thus form stable network compounds with zirconium ions and provide high activity towards phosphorylated peptide. In addition, the hydrophilic pentose (ribose) and purine (adenine) parts in the ATP structure can significantly increase the hydrophilicity of the material and reduce the non-specific adsorption from non-phosphorylated peptides. The results of scanning electron microscopy and elemental analysis indicated that Zr⁴⁺-ATP-MNP was successfully prepared. Using β-casein digest as the sample, 9 phosphopeptides were identified. From digest mixture of β-casein and BSA with molar ratio as low as 1:100, five phosphopeptides were effectively identified with significantly improved signal and the non-phosphorylated peptides were effectively removed. The above results demonstrated the high specificity of Zr⁴⁺-ATP-MNP for both monophosphorylated peptides and multiphosphorylated peptides. The Zr⁴⁺-ATP-MNP was further applied to capture phosphopeptides in milk digest. All these results indicated the promising potential of Zr⁴⁺-ATP-MNP to capture mono- and multi-phosphopeptides in complex biological samples.

Key words:

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1.
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新型锆离子固定化亲和磁纳米粒子的制备及在磷酸化肽高效捕获中的应用

English

Preparation of Novel Zirconium Ion immobilized Affinity Magnetic Nanoparticles and Application in Efficient Capture of Phosphopeptides

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