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1.
本文通过多巴胺自聚合在天然的棉花纤维表面,构建了仿生聚多巴胺(PDA)膜层,然后利用儿茶酚羟基固定Ti~(4+),设计并合成了一种固定金属亲合色谱(Immobilized Metal Ion Affinity Chromatography,IMAC)材料Cotton@PDA-Ti~(4+),并将其用于磷酸化多肽的富集。该材料机械性能好,化学性能稳定和生物相容性好,且制备过程简单,通过简易的In-pipet-tip固相萃取(SPE)装置使整个富集操作过程简便快速。实验结果表明,Cotton@PDA-Ti~(4+)不仅可以从简单的蛋白酶解物(β-casein)中富集磷酸化多肽,并且在含有大量非磷酸化多肽的复杂体系样品中对磷酸化多肽也表现出良好的选择性。另外,利用Cotton@PDA-Ti~(4+)对磷酸化多肽进行富集也有较高的效率。我们将该材料应用于实际样品,如人体血清以及脱脂牛奶酶解物中磷酸化多肽的富集,均表现出了较好的选择性。说明该方法有可能用于磷酸化蛋白质组的全分析。 相似文献
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结合基质辅助激光解吸飞行时间质谱(MALDI-TOF MS)检测技术,考察了Ti-SBA-15介孔材料对β-酪蛋白酶解产物中磷酸化肽的选择性富集性能。实验结果显示,含Ti和Si物质的量比为0.08的Ti-SBA-15介孔材料可选择性地对β-酪蛋白酶解产物中的磷酸化肽进行选择性富集;对于β-酪蛋白和牛血清白蛋白物质的量比为1:100的蛋白质酶解混合液,Ti-SBA-15仍能实现对其磷酸化肽的有效富集。上述结果表明,作为一种多孔、高比表面积的磷酸化多肽的选择性吸附材料,Ti-SBA-15有望在磷酸化蛋白质组的分析中得到广泛的应用。 相似文献
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以精氨酸修饰的磁性微球作为磁性固相萃取(MSPE)平台的载体, 结合基质辅助激光解吸电离飞行时间质谱(MALDI-TOF MS)技术, 实现了对复杂样品中低丰度磷酸化肽的分离富集. 采用场发射扫描电子显微镜、 Zeta电位测定、 红外光谱分析、 振动样品磁强计及X射线衍射分析等手段对合成的功能化磁性材料进行了表征. 选择β-酪蛋白酶解产物磷酸化肽为标准品, 在最佳实验条件下, 利用构建的MSPE-MS平台能够实现对磷酸化肽的高选择性和高灵敏度检测, 检出限为0.1 fmol. 实验结果表明, 经精氨酸修饰的磁性材料对牛奶样品中低含量的磷酸化肽具有较高的选择性, 所建立的方法适用于复杂样品的分离分析. 相似文献
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建立了免疫亲和固相萃取(IAC-SPE)-超高效合相色谱-串联质谱(UPC2-MS/MS)同时测定牛奶中α-玉米赤霉醇、β-玉米赤霉醇、α-玉米赤霉烯醇、β-玉米赤霉烯醇、玉米赤霉烯酮和玉米赤霉酮残留的分析方法。样品用去离子水稀释,经IAC-SPE富集净化后,采用Waters ACQUITY UPC2 Torus 2-PIC色谱柱(50 mm×3.0 mm,1.7 μm)分离,以超临界CO2和0.1%(v/v)甲酸甲醇溶液为流动相,经梯度洗脱后在ESI-模式下检测。经过稀释离心的牛奶样品采用免疫亲和柱净化后没有明显的基质效应,6种目标化合物在1~200 ng/mL范围内线性关系良好,相关系数(r2)≥0.9957;6种目标化合物在3个加标水平下的平均回收率为75.9%~106.5%,日内和日间精密度均≤11.4%。该法专属性好,操作简便,有机溶剂使用量小,与已有的样品测定方法比较更绿色环保,可用于牛奶中α-玉米赤霉醇、β-玉米赤霉醇、α-玉米赤霉烯醇、β-玉米赤霉烯醇、玉米赤霉酮和玉米赤霉烯酮的残留检测。 相似文献
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磷酸化修饰是蛋白质翻译后修饰中最为重要的修饰之一,蛋白质的磷酸化修饰几乎参与生命活动的每一个环节。因此,制备对磷酸化蛋白具有选择性识别性能的材料在磷酸化蛋白质组学中具有重要意义。本实验首先合成胍基离子液体功能单体,通过沉淀聚合法合成聚胍基离子液体材料。通过傅里叶红外光谱(FT-IR)、扫描电子显微镜(SEM)、热重分析仪(TGA)考察了材料的结构、形貌、热稳定性。结果显示所制备材料为粒径约200 nm的球形颗粒。并以标准磷酸化蛋白(β-酪蛋白)为模型蛋白质,考察了聚胍基离子液体材料的识别性能。研究结果表明:材料对磷酸化蛋白具有较高吸附容量(对 β-酪蛋白的最大吸附量达到599.1 mg/g)、较快的吸附速度(1 h内达平衡),而且对磷酸化蛋白表现出较高的选择性。 相似文献
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研究了双酸催化剂不对称催化烯醚和β,γ-不饱和α-酮酸酯的反电子Hetero-Diels-Alder (HDA)反应, 为手性合成3,4-二氢-2H-吡喃类化合物提供了一种新的催化合成方法. InBr3与手性磷酸钙盐Ca(1c)2组合的手性双路易斯酸催化体系能够有效催化3,4-二氢-2H-吡喃和β,γ-不饱和α-酮酸酯的反电子HDA反应, 反应给出优秀的产率(最高达98%), 中等到良好的非对映选择性(最高达89:11)和良好到优秀的对映选择性(最高可达94%). 并且该双酸催化体系也能成功实现其它烯醚(如: 2,3-二氢-2H-呋喃, 乙烯基乙醚)的HDA反应, 获得优秀的非对映选择性(>94:6)和良好的对映选择性. 相似文献
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以标准蛋白质α-酪蛋白的酶解液作为研究对象,考察流动相pH值对磷酸化肽在Click OEG-CD材料上富集选择性的影响。首先以磷酸苯二钠作为模型化合物考察流动相pH值对其在Click OEG-CD材料上的保留影响,结果表明当pH值低于磷酸根的pKa值时,磷酸苯二钠难以电离,与材料的离子交换作用较弱,因而保留也较弱。然后在亲水模式下流动相pH值分别为2, 4, 6时考察Click OEG-CD材料对α-酪蛋白的酶解液中磷酸化肽的富集选择性影响。结果表明,当流动相pH为2时,磷酸化肽不能被材料富集;当pH为4时,磷酸化肽能够被富集,而且洗脱窗口较窄;当pH为6时,磷酸化肽也能够被富集,但是洗脱窗口较宽。因此适合亲水模式下富集磷酸化肽的流动相pH值为4。本研究结果能够为今后将Click OEG-CD材料更好的应用于磷酸化肽富集提供有意义的借鉴。 相似文献
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Highly specific enrichment of phosphopeptides by zirconium dioxide nanoparticles for phosphoproteome analysis 总被引:1,自引:0,他引:1
Large-scale characterization of phosphoproteins requires highly specific methods for the purification of phosphopeptides because of the low abundance of phosphoproteins and substoichiometry of phosphorylation. A phosphopeptide enrichment method using ZrO2 nanoparticles is presented. The high specificity of this approach was demonstrated by the isolation of phosphopeptides from the digests of model phosphoproteins. The strong affinity of ZrO2 nanoparticles to phosphopeptides enables the specific enrichment of phosphopeptides from a complex peptide mixture in which the abundance of phosphopeptides is two orders of magnitude lower than that of nonphosphopeptides. Superior selectivity of ZrO2 nanoparticles for the enrichment of phosphorylated peptides than that of conventional immobilized metal affinity chromatography was observed. Femtomole phosphopeptides from digestion products could be enriched by ZrO2 nanoparticles and can be well detected by MALDI mass spectrometric analysis. ZrO2 nanoparticles were further applied to selectively isolate phosphopeptides from the tryptic digestion of mouse liver lysate for phosphoproteome analysis by nanoliter LC MS/MS (nano-LC-MS/MS) and MS/MS/MS. A total of 248 defining phosphorylation sites and 140 phosphorylated peptides were identified by manual validation using a series of rigid criteria. 相似文献
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We show that BaTiO3 nanoparticles (NPs) can be used as a novel substrate for the rapid enrichment of phosphopeptides from microwave tryptic digests of α-casein and non-fat milk prior to their identification by MALDI-MS. Protein digestion is achieved by microwave tryptic digest for 50?s, and the resulting phosphopeptides can be effectively adsorbed on the surfaces of the NPs. The phosphopeptides were selectively detected via MALDI-MS. Digestion, enrichment and detection are accomplished within ~60?min. The method was applied to the indentification of 24 phosphopeptides from α-casein and of 21 phosphopeptides (of the α-casein type) from nonfat milk. Figure
BaTiO3 NPs as affinity probes for the rapid analysis of phosphopeptides by MALDI MS 相似文献
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Isolation of phosphopeptides using zirconium-chlorophosphonazo chelate-modified silica nanoparticles
Due to the low abundance of phosphoproteins and substoichiometry of phosphorylation, the elucidation of protein phosphorylation requires highly specific materials for isolation of phosphopeptides from biological samples prior to mass spectrometric analysis. In this study, chlorophosphonazo type derivatives of chromotropic acid including p-hydroxychlorophosphonazo (HCPA) and chlorophosphonazo I (CPA I), traditionally used in the photometric determination of transition metal ions, have been employed as chelating ligands in the preparation of novel affinity materials for phosphopeptide enrichment. The chromogenic reagents of HCPA and CPA I were chemically modified on the surface of silica nanoparticles, and the functionalized materials were charged with zirconium ions through the strong complexation between chelating ligands and Zr(4+). The obtained zirconium-chlorophosphonazo chelate-modified silica nanoparticles (Zr-HCPA-SNPs and Zr-CPA I-SNPs) were applied to the selective enrichment of phosphopeptides, followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis. The purification procedures were optimized using α-casein digest at first, and then the performance of these two affinity materials for efficient and specific enrichment of phosphopeptides was evaluated with the tryptic digests of standard proteins (α-casein, β-casein, ovalbumin and bovine serum albumin). It is found that Zr-HCPA-SNPs are superior to Zr-CPA I-SNPs in phosphopeptide enrichment. Using Zr-HCPA-SNPs to trap phosphopeptides in α-casein digest, the detection limit was close to 50fmol based on MALDI-TOF MS analysis. Finally, Zr-HCPA-SNPs were used to directly isolate phosphopeptides from diluted human serum of healthy, diabetes and hypertension persons, respectively. Our results show that the constitution and level of phosphopeptides are remarkably different among the three groups, which indicate the powerful potentials of Zr-HCPA-SNPs in disease diagnosis and biomarker screening. 相似文献
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Phosphopeptides have been isolated and concentrated by use of polyethyleneimine (PEI)-modified magnetic nanoparticles as an
extremely specific affinity probe. The particles specifically captured phosphopeptides from a tryptic digest of a protein
mixture that contained 0.07% (mole/mole) phosphoproteins, which is the highest specificity obtained to date. The time required
for enrichment of the phosphopeptides was 1 min only. PEI-modified magnetic nanoparticles carry positive charges over a wide
range of pH—between 3 and 11. This feature means the particles are effectively dispersed in solution during phosphopeptide
capture. Mass spectrometric analysis revealed the very high efficiency of enrichment of phosphopeptides that contain both
single and multiply-phosphorylated sites. The detection limit in the analysis of phosphopeptides obtained from both bovine
α-casein and β-casein by matrix-assisted laser desorption/ionization mass spectrometry was 5 fmol. This approach was also
used to enrich the phosphopeptides in a protein digest obtained from non-fat milk. 相似文献
15.
Zhao PX Guo XF Wang H Qi CB Xia HS Zhang HS 《Analytical and bioanalytical chemistry》2012,402(3):1041-1056
In this paper, we report, as far as we are aware, the first use of zirconium arsenate-modified silica nanoparticles (ZrAs-SNPs)
for specific capture of phosphopeptides, followed by matrix-assisted laser desorption/ionization mass spectrometric (MALDI
MS) analysis. Under the optimized enrichment conditions, the efficiency and specificity of ZrAs-SNPs were evaluated with tryptic
digests of four standard proteins (α-casein, β-casein, ovalbumin, and bovine serum albumin) and compared with those of titanium
arsenate-modified silica nanoparticles (TiAs-SNPs). The results showed that more selective enrichment of multiply phosphorylated
peptides was observed with ZrAs-SNPs than with TiAs-SNPs whereas TiAs-SNPs resulted in slightly better recovery of singly
phosphorylated peptides. ZrAs-SNPs were chosen for direct capture of phosphopeptides from diluted human serum of healthy and
adenocarcinoma individuals. Our experimental profiling of serum phosphopeptides revealed that the level of phosphorylated
fibrinogen peptide A was up-regulated in the serum of adenocarcinoma patients in comparison with healthy adults. This suggests
the possibility of using ZrAs-SNPs for discovery of biomarkers of the pathogenesis process of tumors. 相似文献
16.
采用液相沉积法(LPD)制备了纳米氧化锆沉积硅胶色谱固定相(ZrO2/SiO2), 并将其应用于亲水作用色谱分离中. 考察并比较了ZrO2/SiO2、 硅胶(SiO2)和氧化锆(ZrO2) 3种色谱固定相在不同有机调节剂比例、 不同pH值及不同盐浓度的流动相条件下的色谱行为. 结果表明, 制备的ZrO2/SiO2色谱柱不仅具有SiO2色谱柱高柱效的优点, 表面沉积的纳米氧化锆还能有效屏蔽硅羟基, 有利于碱性物质的保留和分离, 表现出良好的亲水作用色谱性能. 将ZrO2/SiO2色谱柱用于4种脱氧核苷和5种碱性化合物的分离, 均得到了较好的效果, 展现出其作为色谱固定相良好的应用前景. 相似文献
17.
Nazim Hasan Hui-Fen Wu Yi-Hsien Li Mohd Nawaz 《Analytical and bioanalytical chemistry》2010,396(8):2909-2919
We introduce a simplified sample preparation method using bare TiO2 nanoparticles (NPs) to serve as multifunctional nanoprobes (desalting, accelerating, and affinity probes) for effective enrichment
of phosphopeptides from microwave-assisted tryptic digestion of phosphoproteins (α-casein, β-casein and milk) in Electrospray
Ionization Mass Spectrometry (ESI-MS) and Matrix Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS). The results
demonstrate that TiO2 NPs can effectively enrich and accelerate the digestion reactions of phosphoproteins in aqueous solutions and also from complex
real samples. After the microwave experiments, we directly injected the resulting solutions into the ESI-MS and MALDI-MS systems
for analysis, and excellent sensitivity was achieved without the need for any washing procedure or separation process. The
reasons are attributed to the high binding affinity and selectivity of TiO2 NPs toward phosphopeptides. Thus, phosphopeptides can be adsorbed onto the TiO2 NP surface. The digested or partially digested phosphoproteins can be concentrated onto the TiO2 NP surface. This results in the effective or complete digestion of phosphoproteins in a short period of time (45 s). In addition,
high sensitivity and sequence coverage of phosphopeptide can be obtained using TiO2 NPs as microwave absorbers and affinity probes in MALDI-MS and ESI-MS. This is due to the photocatalytic nature of the TiO2 NPs because the absorption of microwave radiation that can accelerate the activation of trypsin for efficient digestion of
phosphoproteins and enhances the ionization of phosphopeptides. The lowest concentrations detected for ESI-MS and MALDI-MS
were 0.1 μM and 10 fmol, respectively, for α-casein. Comparing the two-step approach of TiO2 NPs with microscale TiO2 particles, the microscale TiO2 particles shows no effect on the microwave-assisted tryptic digestion of phosphoproteins. The current approach offers multiple
advantages, such as great simplicity, high sensitivity and selectivity, straightforward and separation/washing-free technique
for phosphorpeptide enrichment analysis. 相似文献
18.
由ZrO(NO3)2水解得到的ZrO(OH)2水凝胶经碱液回流老化、焙烧后制备了改性ZrO2载体材料,直接浸渍K2RuO4溶液,经还原后用于催化氨合成反应。并运用X射线衍射(XRD)、CO2 程序升温脱附(CO2-TPD)、X射线荧光光谱 (XRF)、N2物理吸附、H2程序升温还原技术(H2-TPR)和CO化学吸附对其进行了表征,重点考察了催化剂性能与载体性能间的构效关系。结果表明,KOH和NH4OH溶液回流均可提高载体的比表面积,但是KOH回流制备的载体同时还具有较强的碱性,因此,负载钌以后表现出最佳活性。在425 ℃、5 MPa、空速为10 000 h-1条件下,出口氨浓度为5.96%,分别较催化剂K-Ru/ZrO2-NH4OH、K-Ru/ZrO2-CP和Ru/ZrO2-NH4OH提高了11%、143%和103%。与活性组分分散度相比,载体碱性强度对活性的促进作用更为明显。 相似文献
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We have developed nanoparticle-modified monoliths in pipette tips for selective and efficient enrichment of phosphopeptides. The 5 μL monolithic beds were prepared by UV-initiated polymerization in 200 μL polypropylene pipette tips and either iron oxide or hydroxyapatite nanoparticles were used for monolith modification. Iron oxide nanoparticles were prepared by a co-precipitation method and stabilized by citrate ions. A stable coating of iron oxide nanoparticles on the pore surface of the monolith was obtained via multivalent electrostatic interactions of citrate ions on the surface of nanoparticles with a quaternary amine functionalized poly(2-hydroxyethyl methacrylate-co-ethylene dimethacrylate) monolith. Hydroxyapatite nanoparticles were incorporated into the poly(2-hydroxyethyl methacrylate-co-ethylene dimethacrylate) monolith by simply admixing them in the polymerization mixture followed by in situ polymerization. The nanoparticle-modified monoliths were compared with commercially available titanium dioxide pipette tips. Performance of the developed and commercially available sorbents was demonstrated with the efficient and selective enrichment of phosphopeptides from peptide mixtures of α-casein and β-casein digests followed by off-line MALDI/MS analysis. 相似文献