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1.
余琼卫  方凯敏  何小梅  郑杰  冯钰锜 《色谱》2018,36(3):237-244
采用液相沉积法在棉花纤维的表面成功沉积了纳米氧化锆颗粒,并将其装填在移液枪的吸头内,通过移液枪的抽吸实现对磷酸化多肽的萃取,萃取过程只需要2 min,方法简单、快速。该材料不仅可以从β-酪蛋白酶解物这种简单的体系中萃取出9个磷酸化多肽,还可以从物质的量比为1∶100的β-酪蛋白酶解物和牛血清白蛋白(BSA)酶解物混合物这类含有大量非磷酸化多肽的复杂样品中萃取出4种磷酸化多肽,且没有非磷酸化多肽被检出,表现出较好的萃取选择性。将该材料应用于人血清和脱脂牛奶这两种复杂实际样品的酶解物中磷酸化多肽的快速富集萃取,分别检测出5种和9种磷酸化多肽,均表现出较好的选择性。  相似文献   

2.
以0.1μmol/Lβ-casein磷酸化蛋白酶解液为对象,利用在酸性条件下对PO43-能够特异性吸附的氧化铁材料为新载体,对介孔氧化铁材料富集分离磷酸化肽段的孵育液酸含量、孵育液有机溶剂含量和洗脱液选择的条件进行了优化,结果表明:室温条件下,在含有0.1%乙酸和30%乙腈的孵育液中孵育5min后,经1mol/LNH3.H2O溶液的洗脱,介孔氧化铁可有效地将磷酸化肽段从蛋白酶解液中富集分离。本方法也可以选择性地提取α-casein磷酸化蛋白,实现了简单、快速、高效的磷酸化肽段和蛋白的富集分离。同时,通过MALDI-TOF串级质谱分析,成功地完成了在优化条件下分离出的磷酸化肽段磷酸位点的鉴定。  相似文献   

3.
蛋白质的磷酸化修饰广泛存在于细胞生命活动进程中,在细胞的分化、繁殖、代谢及信号转导等方面均发挥着重要作用.MALDI-TOF-MS技术因其高灵敏度等优点,已成为蛋白质研究的重要分析工具之一.由于磷酸化蛋白的天然丰度和离子化效率较低、检测信号被抑制等缺点,因此需要发展新的分析方法以提高其分析灵敏度和选择性.本文建立了基于介孔氧化钛的多磷酸化肽段高效分析方法,同时利用了TiO2对磷酸化肽的选择性吸附作用和介孔材料特殊空间结构所形成的对多磷酸肽的动力学及热力学吸附效应,结合MALDI-TOF-MS技术,成功地实现了对混合溶液中多磷酸化肽的选择性富集和高灵敏度分析鉴定,与其他方法相比,分析性能得到明显的提高,可实现对低量磷酸化肽段的识别与检测.  相似文献   

4.
周丝  彭宇  陈燕萍  贾琼 《分析化学》2023,(6):1051-1060
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。  相似文献   

5.
叶明亮 《色谱》2013,31(1):1-3
<正>1磷酸肽富集的新材料与新机理磷酸肽的选择性富集是蛋白质组磷酸化分析是否成功的关键步骤。金属氧化物亲和色谱是富集磷酸肽的一类重要方法,一系列的过渡金属氧化物(如二氧化钛、二氧化锆等)被用于磷酸钛的选择性富集。最近,表面涂有三氧化二镧的硅胶复合材料被用于磷酸肽的富集。Jabeen等利用标准蛋白质酶解液考察了该方法的富集效果,发现在两个标准磷酸化蛋白质α-酪蛋白和β-酪蛋白与非磷酸化蛋白质牛血清白蛋白的物质的量比为1∶1∶100时仍能检测到3个磷酸肽,说明该方法有较好的富集  相似文献   

6.
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   

7.
制备了3种内外表面修饰不同官能团的硅基介孔材料,采用傅立叶变换红外光谱对修饰后材料的官能团进行了表征,并采用高效液相色谱对材料选择性富集低分子量蛋白质的能力进行了考查。3种材料对低分子量蛋白质和小肽均具有选择性富集能力,其中乙烯-二醇材料的富集能力最好。采用乙烯-二醇介孔材料装填预柱,考察了其对低分子量蛋白质的在线富集能力。高分子量蛋白质(卵清蛋白,分子量43000Da)在该预柱上没有保留,在死时间里直接流出柱外,而低分子量蛋白质(胰岛素,分子量5700Da)在该预柱上得到了较好的选择性萃取。保留在柱上的低分子量样品通过提高流动相中的有机相含量进行洗脱。研究工作对低分子量蛋白质和小肽的在线富集提供了一种新技术。  相似文献   

8.
基于碱性磷酸酶的去磷酸化作用,发展了一种可改善多磷酸化肽电喷雾质谱检测效果的技术。将β-酪蛋白酶解产物用TiO2柱富集后,用碱性磷酸酶进行处理,并经微柱液相色谱分离后采用串联质谱进行鉴定。通过谱图中存在相对分子质量与根据氨基酸序列预计的单磷酸化肽相差80 的色谱峰,可以证实样品中含有单磷酸化肽。此外,经碱性磷酸酶处理后的样品的色谱峰数目的增加,说明样品中可能存在多磷酸化肽段。通过控制去磷酸化反应的程度,使四磷酸化肽的部分磷酸基团被去除,从而可以推断其中3 个磷酸化位点可能处于氨基酸残基序列的第17、18和19位。  相似文献   

9.
蛋白质磷酸化是最为广泛的翻译后修饰之一。在生物体液或组织中,许多低丰度的磷酸化蛋白和磷酸化肽是具有高度临床灵敏性和特异性的生物标记物,这些生物分子对许多疾病的检测和病理的阐释可能提供重要的信息。因为蛋白质磷酸化动态可逆且磷酸化蛋白丰度很低,所以很难直接从复杂的生物样品中直接检测到磷酸化蛋白和磷酸化肽。纳米结构材料因其大比表面积、丰富的活性亲合位点和特殊结构,在磷酸化肽和磷酸化蛋白的分离和富集方面已经引起了特别的关注,并成为目前磷酸化蛋白质组学富集和鉴定方面的研究热点。许多介孔、磁性、杂化或化学修饰的亲合材料被研发并用于磷酸化蛋白/多肽的富集与分离;此外,一些多功能纳米结构材料也被研发并用于蛋白质组学中磷酸化蛋白/多肽的快速高效的富集提纯。在这篇综述中,我们专注于纳米结构材料在磷酸化蛋白/多肽富集和提纯方面的最新进展。  相似文献   

10.
采用溶胶-凝胶法制备了氧化硅-氧化锆复合氧化物(SiO2-ZrO2),并对其物理与化学性质进行了详细地表征.以α-酪蛋白的酶解产物为探针,比较了氧化硅-氧化锆复合氧化物与氧化锆对磷酸化肽段的富集能力.结果表明: 在pH<2时富集,SiO2-ZrO2对磷酸化肽的选择性要优于ZrO2;使用SiO2-ZrO2材料时,多磷酸化肽段的回收率更高.结合MALDI-TOF MS检测技术,SiO2-ZrO2成功地应用于脱脂牛奶酶解产物中磷酸化肽的分离和富集.  相似文献   

11.
Extraction of phosphopeptides from rather complex biological samples has been a tough issue for deep and comprehensive investigation into phosphoproteomes. In this paper, we present a series of Ti-doped mesoporous silica (Ti-MPS) materials with tunable composition and controllable morphology for highly efficient enrichment of phosphopeptides. By altering the molar ratio of silicon to titanium (Si/Ti) in the precursor, the external morphology, Ti content, internal long-rang order, and surface area of Ti-MPS were all modulated accordingly with certain regularity. Tryptic digests of standard phosphoprotein α- and β-casein were employed to assess the phosphopeptide enrichment capability of Ti-MPS series. At the Si/Ti molar ratio of 8:1, the optimum enrichment performance with admirable sensitivity and capacity was achieved. The detection limit for β-casein could reach 10 fmol, and 15 phosphopeptides from the digest of α-casein were resolved in the spectrum after enrichment, both superior to the behavior of commercial TiO2 materials. More significantly, for the digest of human placenta mitochondria, 396 phosphopeptides and 298 phosphoproteins were definitely detected and identified after enrichment with optimized Ti-MPS material, demonstrating its remarkable applicability for untouched phosphoproteomes. In addition, this research also opened up a universal pathway to construct a composition-tunable functional material in pursuit of the maximum performance in applications.
Figure
From human placenta mitochondria to MS  相似文献   

12.
水热一步法合成Ti-SBA-15分子筛及其催化性能研究   总被引:15,自引:1,他引:14  
采用钛酸丁酯和乙酰丙酮作用后的产物作为钛的前驱体,水热法一步合成出了 Ti-SBA-15分子筛。通过X射线衍射、N2气吸附脱附、紫外—可见漫反射等表征手段 ,证明在Si/Ti=50,25,20时,钛原子成功地取代了SBA-15中的硅原子而没有改 变SBA-15高度有序的介孔二维六角结构,其中钛全部以四配位状态存在,而且高度 分散。当Si/Ti=12.5时,钛的分散度降低,部分钛聚集生成二氧化钛。与用两步 法合成的Ti-SBA-15比较,一步法合成的Ti-SBA-15中钛分散度好,添加量高,对催 化氧化环己烯有较高的催化活性。对一步法和两步法合成Ti-SBA-15的机理以及相 应产物在结构以及催化活性上的差异进行了探讨。  相似文献   

13.
This study presented an approach to prepare monodisperse immobilized Ti4+ affinity chromatography (Ti4+-IMAC) microspheres for specific enrichment of phosphopeptides in phosphoproteome analysis. Monodisperse polystyrene seed microspheres with a diameter of ca. 4.8 μm were first prepared by a dispersion polymerization method. Monodisperse microspheres with a diameter of ca. 13 μm were prepared using the seed microspheres by a single-step swelling and polymerization method. Ti4+ ion was immobilized after chemical modification of the microspheres with phosphonate groups. The specificity of the Ti4+-IMAC microspheres to phosphopeptides was demonstrated by selective enrichment of phosphopeptides from mixture of tryptic digests of α-casein and bovine serum albumin (BSA) at molar ratio of 1 to 500 by MALDI-TOF MS analysis. The sensitivity of detection for phosphopeptides determined by MALDI-TOF MS was as low as 5 fmol for standard tryptic digest of β-casein. The Ti4+-IMAC microspheres were compared with commercial Fe3+-IMAC adsorbent and homemade Zr4+-IMAC microspheres for enrichment of phosphopeptides. The phosphopeptides and non-phosphopeptides identified by Fe3+-IMAC, Zr4+-IMAC and Ti4+-IMAC methods were 26, 114, 127 and 181, 11, 11 respectively for the same tryptic digest samples. The results indicated that the Ti4+-IMAC had the best performance for enrichment of phosphopeptides.  相似文献   

14.
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.  相似文献   

15.
Shiau KJ  Hung SU  Lee HW  Wu CC 《The Analyst》2011,136(9):1922-1927
Simultaneous detection of multiply and singly phosphorylated peptides using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is challenging because of suppression effects during ionization. In oder to overcome this problem, this study presents a new approach to improve the detection of phosphopeptides by stepwise enrichment using polyarginine-coated (PA-coated) and titanium dioxide-coated (TiO(2)-coated) nanodiamonds for fractionation of multiply and singly phosphorylated peptides prior to on-probe MALDI MS analysis. The feasibility of this approach was demonstrated using synthetic peptides containing different numbers of phosphate groups, tryptic digests of α-casein, β-casein, and complex protein mixtures. The high specificity of the approach is shown in its effective enrichment and fractionation of phosphopeptides from the digest of β-casein and bovine serum albumin at a molar ratio as low as 1 : 1000, which out-performs the commercial Fe(3+)-IMAC and TiO(2) isolation kits. It offers a simple and effective alternative for the fractionation and identification of multiply and singly phosphorylated peptides by MALDI MS and allows for deduction of more information from limited starting materials.  相似文献   

16.
Novel Fe(3)O(4)@La(x)Si(y)O(5) affinity microspheres consisting of a superparamagnetic Fe(3)O(4) core and an amorphous lanthanum silicate shell have been synthesized. The core-shell-structured Fe(3)O(4)@La(x)Si(y)O(5) microspheres, with a mean size of ca. 480 nm, had rough lanthanum silicate surfaces and displayed relatively strong magnetism (47.2 emu g(-1)). This novel affinity material can be used for selective capture, rapid magnetic separation, and part dephosphorylation (which plays an important role in identifying phosphopeptides in MS) of the phosphopeptides in a peptide mixture. Its ability to selectively trap and magnetically isolate as well as label the phosphopeptides was evaluated using a standard phosphorylated protein (β-casein) and a real sample (human serum). Phosphopeptides and their corresponding label ions were detected for concentrations of β-casein as low as 1 × 10(-9) M and in mixtures of β-casein and BSA with molar ratios as low as 1:50. In addition, this affinity material, with its labeling properties, is superior to commercial TiO(2) beads in terms of interference from non-phosphopeptide molecules. These results reveal that the lanthanum silicate coated magnetic microspheres represent a promising affinity material for the rapid purification and recognition of phosphopeptides.  相似文献   

17.
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   

18.
A zirconium(IV)-based metal organic framework (Zr-MOF) was deposited on polydopamine-coated silica microspheres to form microspheres of type SiO2@PDA@Zr-MOF. These were packed into capillary columns for enrichment of phosphopeptides. The column was off-line coupled to both matrix-assisted laser desorption/ionization time of flight mass spectrometry and LC-ESI-MS/MS. The method has a detection limit as low as 4 fmol of β-casein digest and a selectivity as high as 1:1000 (molar ratio of β-casein and BSA digest). It was applied to the analysis of human saliva. In total, 240 endogenous phosphopeptides were identified in only 25 μL human saliva.
Graphical abstract A zirconium-based metal organic framework (Zr-MOF) was modified outside of polydopamine-coated silica microspheres to form microspheres named SiO2@PDA@Zr-MOF. Then they were packed in capillary columns for selective enrichment of phosphopeptides via interaction between Zr-O clusters and phosphate groups. The pre-concentration resulted in a better detection of phosphopeptides by mass spectrometry. Tris: Tris(hydroxymethyl)aminomethane; DMF: Dimethyl Formamide; Zr-MOF: Zirconium(IV)-organic framework; MOAC: Metal oxide affinity chromatography.
  相似文献   

19.
以介孔分子筛SBA-15为载体,Ni组分采用柠檬酸(CA)配合法,制备了Ni2P质量含量为25%~45%、P/Ni为0.8、CA/Ni为0~1.5的一系列CA-Ni2P/SBA-15催化剂.利用XRD和N2吸脱附表征了催化剂结构,以二苯并噻吩(DBT)为模型硫合物,对催化剂加氢脱硫(HDS)性能进行了评价,考察了CA/Ni比对催化剂结构和反应性能的影响.结果表明,催化剂仍然保持有介孔结构,催化活性物相为Ni2P.反应温度为300~340℃时,Ni2P含量和CA/Ni比都对催化剂的性能有一定的影响,反应温度在360℃以上时,Ni2P含量和CA/Ni比对催化剂性能的影响不明显.Ni2P含量为35%、CA/Ni比为1.0的催化剂具有最好的HDS活性,DBT的转化率可达98%.  相似文献   

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