季铵化磁性壳聚糖复合材料对磷酸化肽的富集研究

蛋白质磷酸化是最重要和最普遍的翻译后修饰之一,蛋白质磷酸化的测定对于全面了解生物过程中的磷酸化途径至关重要。质谱技术是分析蛋白质磷酸化的重要手段,但磷酸化肽固有的低丰度、低电离效率以及与非磷酸化肽共存等特性严重影响质谱对其进行直接分析。为解决此问题,需在质谱分析前对磷酸化肽进行选择性富集。本研究制备了一种基于季铵化磁性壳聚糖的复合材料用于磷酸化肽的富集。此磁性材料具有快速的磁响应性、良好的生物相容性、正电性以及廉价易得等优点。采用β-酪蛋白作为模型蛋白质,结果表明此材料对磷酸化肽具有良好的富集选择性。经过富集后,结合基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS）检测手段,方法的检出限为0.4 fmol。本方法被成功用于脱脂牛奶中磷酸化肽的检测,表明其对复杂样品中磷酸化肽的富集和检测具有良好的应用潜力。     

功能化磁性纳米材料在磷酸化肽富集中的应用

蛋白质磷酸化是最重要和最普遍的翻译后修饰之一。基于质谱的技术已成为分析蛋白质磷酸化的重要手段。然而,磷酸化肽固有的低丰度和电离效率以及由非磷酸化肽共存引起的严重抑制使得直接质谱分析仍然是一个挑战。为解决此问题,需在质谱分析前对磷酸化蛋白质进行选择性富集。磁性纳米材料具有良好的磁响应性,可以在外界磁铁的帮助下实现与溶液的迅速分离。功能化磁性纳米材料作为一种新型的分析技术已在蛋白质组学研究中得到广泛的应用。该文就近年来对磁性纳米粒子进行各种功能化修饰以提高其特异性吸附能力的吸附材料在磷酸化肽的富集方面的应用予以综述,并展望了功能化磁性纳米材料在磷酸化肽富集领域的应用前景。     

多金属氧酸盐/壳聚糖磁性复合材料的制备及其用于磷酸化肽的富集

建立了一种基于多金属氧酸盐磁性材料富集磷酸化肽的方法。采用层层自组装技术制备多金属氧酸盐/壳聚糖磁性材料,结合基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS）检测手段,用于磷酸化肽的富集。该磁性材料具有快速磁响应、亲水性、正电性等优点,对磷酸化肽具有高的富集选择性。实验用β-酪蛋白作为模型蛋白质,通过富集后,方法的检出限为0.02 fmol,说明合成的磁性材料对微量蛋白样品分析具有很高的应用潜力。     

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

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...     

迷迭香酸磁性分子印迹聚合物的合成及应用

本文使用磁性分子印迹技术,合成了同时具有超顺磁性和吸附选择性的迷迭香酸磁性分子印迹聚合物(R-MMIPs)。在外加磁场的作用下,R-MMIPs可以快速提取分离复杂基质中的迷迭香酸。采用扫描电子显微镜(SEM)、傅里叶变换红外光谱(FT-IR)、透射电子显微镜(TEM)和磁强振动计(VSM)对纳米材料的微观结构进行表征。通过吸附实验评价其结合能力和特异性,其最大吸附量为8.68 mg/g。此外,R-MMIPs成功地实现了从实际样品中提取和分离迷迭香酸,加标回收率为88.1%～107.5%。本方法具有操作简便、吸附速度快、选择性高等优点,适用于复杂基质中迷迭香酸的快速选择性分离。     

离子液体基磁性固相萃取技术的研究进展

磁性固相萃取(Magnetic solid-phase extraction,MSPE)是一种采用磁性材料作为吸附剂的新型样品前处理技术,发展新型的磁性材料作为吸附剂是MSPE领域的研究热点。用离子液体(Ionic liquid,IL)修饰磁性材料作为吸附剂既具有MSPE操作简单、萃取快速、基质干扰小的优点,又兼具IL结构的可设计性和易功能化的优点,在样品前处理领域引起了广泛关注。该文综述了IL修饰磁性吸附剂的制备方法(物理涂敷、化学键合和包埋法),IL-MSPE的萃取方式(传统的MSPE,混合半胶束-MSPE和分散液液微萃取-MSPE),以及IL-MSPE在有机污染物、金属离子和生物活性物质萃取分离中的应用,并对该技术的发展趋势进行了展望。     

磁性固相萃取技术研究的新进展

基于磁性材料的分离技术是近年来国内外研究的一个热点领域,该技术在细胞分离、药物转运、酶的固定化、催化、吸附-分离、材料科学、环境等诸多领域中都展示了应用前景。磁性纳米材料可作为磁性固相萃取(MSPE)的吸附剂。与常规的固相萃取(SPE)柱填料相比,纳米粒子的比表面积大、扩散距离短,只需使用少量的吸附剂和较短的平衡时间就能实现萃取分离,因此具有较高的萃取能力和萃取效率。经功能化修饰,磁性吸附剂有望实现对分析物的选择性萃取。另外,磁性吸附剂经适当的润洗之后可以循环使用。MSPE仅通过施加一个外部磁场即可实现相分离,因此操作简单、省时快速、无需离心过滤等繁琐操作,避免了传统SPE吸附剂需装柱和样品上样等耗时问题,而且在处理生物、环境样品时不会存在SPE中遇到的柱堵塞的问题。MSPE技术在痕量污染物萃取分离领域已得到了应用［1,2］。本文对MSPE技术研究的最新进展做一简要介绍。     

新型反相/强阴离子交换混合模式材料用于磷酸化肽富集

建立了新型反相/强阴离子交换混合模式材料(C18/SAX)的磷酸化肽富集方法.考察了流动相组成(乙腈浓度、甲酸浓度、缓冲盐浓度)对酪蛋白(α-Casein)酶解液中磷酸化肽分离选择性的影响.实验结果表明,磷酸化肽在C18/SAX上的保留行为受疏水和离子交换作用力的共同调控,单磷酸化肽先于多磷酸化肽从材料上洗脱出来.随着甲酸浓度增加,磷酸化肽的保留减弱;随着盐浓度增加,磷酸化肽保留变小.采用优化后的流动相,建立以20％ ACN/20 mmol/L NH4Ac作为上样溶液,20％ ACN/0.1％ FA和50％ ACN/100 mmol/L NH4Ac/2％ FA分别作为洗脱液分段洗脱单、多磷酸化肽的方法.以α-Casein和人血清白蛋白(HSA)酶解液的混合溶液(1∶20,n/n)作为模拟样品,实现了单、多磷酸化肽的同时富集和分段洗脱,分别检测到4条单磷酸化肽和14条多磷酸化肽的信号.将本方法用于牛奶中的磷酸化肽检测,共鉴定到4条单磷酸化肽和8条多磷酸化肽信号.结果表明,本富集方法选择性高,有良好的应用前景.     

万古霉素修饰磁性纳米粒子的制备及其细菌分离功能

制备了万古霉素修饰的磁性纳米粒子, 并研究了其对金黄色葡萄球菌(S.aureus)和大肠杆菌(E.coli BL21)的选择性吸附分离特性.     

基于硼酸基功能化的磁性表面印迹聚合物制备及其对目标糖蛋白的识别

以硼酸基功能化的磁性Fe3O4纳米粒子(Fe3O4NPs)为载体,卵清蛋白(OB,一种糖蛋白)作模板分子,多巴胺(DA)为单体,采用表面印迹的方法,制备了一种磁性表面分子印迹聚合物纳米粒子。用平衡吸附实验研究了其吸附性能和识别选择性。结果表明,该分子印迹聚合物对目标糖蛋白(OB)具有较高的选择性和吸附量,并且具有良好的磁性,有利于进行快捷的磁性分离,这将为复杂生物样品中目标糖蛋白的专一性识别提供一种新的途径。     

The highly selective capture of phosphopeptides by zirconium phosphonate-modified magnetic nanoparticles for phosphoproteome analysis

The highly selective capture of phosphopeptides from proteolytic digests is a great challenge for the identification of phosphoproteins by mass spectrometry. In this work, the zirconium phosphonate-modified magnetic Fe₃O₄/SiO₂ core/shell nanoparticles have been synthesized and successfully applied for the selective capture of phosphopeptides from complex tryptic digests of proteins before the analysis of MALDI-TOF mass spectrometry with the desired convenience of sample handling. The ratio of magnetic nanoparticle to protein and the incubation time for capturing phosphopeptides from complex proteolytic digests were investigated, and the optimized nanoparticle-to-protein ratio and incubation time were between 15:1 to 30:1 and 30 min, respectively. The excellent detection limit of 0.5 fmol β-casein has been achieved by MALDI-TOF mass spectrometry with the specific capture of zirconium phosphonate-modified magnetic Fe₃O₄ nanoparticles. The great specificity of zirconium phosphonate-modified magnetic Fe₃O₄ nanoparticles to phosphopeptides was demonstrated by the selective capture of phosphopeptides from a complex tryptic digest of the mixture of α-casein and bovine serum albumin at molar ratio of 1 to 100 in MALDI-TOF-MS analysis. An application of the magnetic nanoparticles to selective capture phosphopeptides from a tryptic digest of mouse liver lysate was further carried out by combining with nano-LC-MS/MS and MS/MS/MS analyses, and a total of 194 unique phosphopeptides were successfully identified.     

Preparation of magnetic hydroxyapatite clusters and their application in the enrichment of phosphopeptides

A novel strategy for the effective enrichment of phosphopeptides based on magnetic hydro‐xyapatite (HAp) clusters was developed in the current study. The structure of HAp ensures its probable separation capability, including cation exchange with P‐sites (negatively charged pairs of crystal phosphates), calcium coordination, anion exchange with C‐sites (positively charged pairs of crystal calcium ions). The prepared magnetic HAp clusters showed good performance on the efficient enrichment of phosphopeptides from the digestion mixture of β‐casein and BSA. Compared to commercial HAp particles, the magnetic HAp clusters exhibited better selectivity toward phosphopeptides. In addition, the use of magnetic material greatly simplified the enrichment procedure, which avoided the tedious centrifugation steps in a typical phosphopeptides enrichment protocol. Finally, the material was successfully applied in the enrichment of phosphopeptides from human serum. Taken together, the efficient enrichment of the phosphopeptides by the easily prepared magnetic HAp clusters demonstrated a rapid and convenient strategy for the purification of phosphopeptides from complex samples, which may facilitate protein phosphorylation studies.     

Enrichment of phosphopeptides using bare magnetic particles

Magnetic iron(II, III) oxide (magnetite, Fe(3)O(4)) nanoparticles were used to selectively enrich phosphopeptides from tryptic digests of bovine beta-casein and from tryptic digest mixtures containing bovine beta-casein, cytochrome c, bovine serum albumin, and horse heart myoglobin. The magnetic property of the particles permits an easy and speedy enrichment process. No enrichment of phosphopeptides was observed from ferric magnetic iron(III) oxide (Fe(2)O(3)) nanoparticles. These data collectively demonstrate that the enrichment of phosphopeptides using magnetic iron(II, III) oxide nanoparticles is a practical method for the selective analysis of phosphopeptides and could be helpful in isolating and analyzing phosphorylated peptides from complex biological samples.     

Facile synthesis of gallium ions immobilized and adenosine functionalized magnetic nanoparticles with high selectivity for multi-phosphopeptides

Despite recent advances in phosphoproteome research, detection and characterization of multi-phosphopeptides have remained a challenge. Here we present a novel IMAC strategy for effective extracting multi-phosphopeptides from complex samples, through Ga³⁺ chelation to the adenosine tri-phosphate (ATP)-functionalized magnetic nanoparticles (Ga³⁺-ATP-MNPs). The high specificity of Ga³⁺-ATP-MNPs was demonstrated by efficient enriching multi-phosphopeptides from the digest mixture of β-casein and BSA with molar ratio as low as 1:5000. Ga³⁺-ATP-MNPs were also successfully applied for the phosphoproteome analysis of rat liver mitochondria, resulting in the identification of 193 phosphopeptides with 331 phosphorylation sites from 158 phosphoproteins. In other words, 54.4% of the phosphopeptides trapped by Ga³⁺-ATP-MNPs were observed with more than one phosphorylated sites, resulting in significant improvement on the identification of peptides with multi-phosphorylated sites. The high specificity of Ga³⁺-ATP-MNPs towards multi-phosphopeptides may be due to the synergistic effect of the strong hydrophilic surface functionalized by ATP and the proper chelating strength provided by Ga³⁺. Moreover, the unique magnetic core of Ga³⁺-ATP-MNPs also facilitates the isolation process and on-plate enrichment for direct MALDI MS analysis with limit of detection as low as 30 amol. This new affinity-based protocol is expected to provide a powerful approach for characterizing multiple phosphorylation sites on proteins in complex and dilute analytes, which may be explored as complementary technique for improving the coverage of phosphoproteome.     

过渡金属掺杂磁性纳米粒子在生物医学领域中的研究进展

在过去50多年中,磁性纳米粒子(MNPs)由于其可协调的磁性、非侵入性、易操控性和良好的生物相容性等优点得到了广泛的关注.从具有复合结构或不同形状的MNPs的合成方法到与MNPs相关的大量表征技术,其应用领域也与我们的生活紧密相关.然而,MNPs的复杂磁行为受到多种参量的影响,包括粒径、成分、形状和结构等.基于此,通过...     

Iron oxide/tantalum oxide core–shell magnetic nanoparticle-based microwave-assisted extraction for phosphopeptide enrichment from complex samples for MALDI MS analysis

A new type of metal-oxide-coated magnetic nanoparticles (NPs)—tantalum-oxide-coated magnetic iron oxide (Fe₃O₄@Ta₂O₅) NPs—which are used as affinity probes for selectively trapping phosphopeptides from complex samples, is demonstrated in this study. In this approach, phosphopeptide enrichment was achieved by incubating the NPs with sample solutions under microwave heating within 1 min. The NP–target species conjugates were readily isolated from samples by magnetic separation followed by matrix-assisted laser desorption/ionization (MALDI) mass spectrometric analysis. When using human serum as the sample, phosphorylated fibrinopeptide-A-derived ions are the only ions observed in the MALDI mass spectra after enrichment by the Fe₃O₄@Ta₂O₅ NPs. Furthermore, only phosphopeptides appear in the MALDI mass spectra after using the affinity probes to selectively trap target species from the tryptic digest of a cell lysate and milk sample. The results demonstrated that the Fe₃O₄@Ta₂O₅ NPs have the capability of selectively trapping phosphorylated peptides from complex samples. The detection limit of this approach for a phosphopeptide (FQpSEEQQQTEDELQDK) was ~10 fmol. Figure For the first time, tantalum oxide-coated magnetic iron oxide (Fe₃O₄@Ta₂O₅) NPs were demonstrated as suitable affinity-probes for selectively trapping phosphopeptides from complex samples. To shorten the analysis time, phosphopeptide enrichment was achieved by incubating the NPs with sample solutions under microwave-heating within 1 min. MALDI MS was employed for characterization of the species trapped by the NPs.     

葡萄糖-6-磷酸功能化亲水磁探针：有效分离富集糖肽/磷酸肽的双用途亲和材料

在Fe₃O₄磁芯上通过逐层修饰构建了葡萄糖-6-磷酸(G6P)功能化亲水磁探针Fe₃O₄@PDA@TiO₂@G6P. 聚多巴胺(PDA)可以作为偶联连接剂进一步接枝二氧化钛(TiO₂); 接枝的TiO₂除作为G6P的锚定位点外, 还 可通过金属氧化物亲和层析技术有效富集磷酸肽; G6P的官能化赋予了纳米球高亲水性的表面, 并利用亲水作用液相色谱法实现了糖肽的捕捉. 实验结果表明, 探针对糖肽具有低的检出限(0.1 fmol/μL)、 高的选择性 [m(HRP)∶m(BSA)=1∶1000]、 良好的重复性(10次循环)和高的负载量(300 mg/g); 对磷酸肽具有低的检出限(0.02 fmol/μL)和高的选择性[n(β-casein)∶n(BSA)=1∶1000). 此外, 这种双用途亲和材料具有同时富集糖肽和磷酸肽的能力,可从人唾液中鉴定出34个糖肽和36个磷酸肽, 表明其在多种翻译后修饰的蛋白质组学分析中具有巨大的应用前景.     

Zirconium arsenate-modified magnetic nanoparticles: preparation, characterization and application to the enrichment of phosphopeptides

Phosphorylation, one of the most important post-translational modifications of protein, plays a crucial role in a large number of biological processes. Large-scale identification of protein phosphorylation by mass spectrometry is still a challenging task because of the low abundance of phosphopeptides and sub-stoichiometry of phosphorylation. In this work, a novel strategy based on the specific affinity of zirconium arsenate to the phosphate group has been developed for the effective enrichment of phosphopeptides. Zirconium arsenate-modified magnetic nanoparticles (ZrAs-Fe(3)O(4)@SiO(2)) were prepared by covalent immobilization of zirconium arsenate on Fe(3)O(4)@SiO(2) magnetic nanoparticles under mild conditions, and characterized by transmission electron microscope (TEM), Fourier transform infrared (FT-IR) spectroscopy, energy dispersive X-ray spectroscopy (EDX) and vibrating sample magnetometer (VSM). The prepared ZrAs-Fe(3)O(4)@SiO(2) was applied for the selective enrichment of phosphopeptides from the digestion mixture of phosphoproteins and bovine serum albumin (BSA). Our results demonstrated that the ZrAs-Fe(3)O(4)@SiO(2) magnetic nanoparticles possess higher selectivity for phosphopeptides and better capture capability towards multiply-phosphorylated peptides than commercial zirconium dioxide (ZrO(2)), which has been widely employed for the enrichment of phosphopeptides. In addition, endogenous phosphopeptides from human serum can be effectively captured by ZrAs-Fe(3)O(4)@SiO(2) magnetic nanoparticles. It is the first report, to the best of our knowledge, in which the zirconium arsenate-modified magnetic nanoparticles were successfully applied to the enrichment of phosphopeptides, which offers the potential application of this new material in phosphoproteomics study.     

Hydrophilic Nb-immobilized magnetic core–shell microsphere – A novel immobilized metal ion affinity chromatography material for highly selective enrichment of phosphopeptides

Rapid and selective enrichment of phosphopeptides from complex biological samples is essential and challenging in phosphorylated proteomics. In this work, for the first time, niobium ions were directly immobilized on the surface of polydopamine-coated magnetic microspheres through a facile and effective synthetic route. The Fe₃O₄@polydopamine-Nb⁵⁺ (denoted as Fe₃O₄@PD-Nb⁵⁺) microspheres possess merits of high hydrophilicity and good biological compatibility, and demonstrated low limit of detection (2 fmol). The selectivity was also basically satisfactory (β-casein:BSA = 1:500) to capture phosphopeptides. They were also successfully applied for enrichment of phosphopeptides from real biological samples such as human serum and nonfat milk. Compared with Fe₃O₄@PD-Ti⁴⁺ microspheres, the Fe₃O₄@PD-Nb⁵⁺ microspheres exhibit superior selectivity to multi-phosphorylated peptides, and thus may be complementary to the conventional IMAC materials.