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.     

Ti (IV) attached‐phosphonic acid functionalized capillary monolith as a stationary phase for in‐syringe‐type fast and robust enrichment of phosphopeptides

In this study, poly(vinylphosphonic acid‐co‐ethylene dimethacrylate), poly(VPA‐co‐EDMA) capillary monolith was synthesized as a starting material for obtaining a stationary phase for microscale enrichment of phosphopeptides. The chelation of active phosphonate groups with Ti (IV) ions gave a macroporous monolithic column with a mean pore size of 5.4 μm. The phosphopeptides from different sources were enriched on Ti (IV)‐attached poly(VPA‐co‐EDMA) monolith using a syringe‐pump. The monolithic capillary columns exhibited highly sensitive/selective enrichment performance with phosphoprotein concentrations as low as 1.0 fmol/mL. Six different phosphopeptides were detected with high intensity by the treatment of β‐casein digest with the concentration of 1.0 fmol/mL, using Ti (IV)@poly(VPA‐co‐EDMA) monolith. Highly selective enrichment of phosphopeptides was also successfully carried out even at trace amounts, in a complex mixture of digested proteins (molar ratio of β‐casein to bovine serum albumin, 1:1500) and three phosphopeptides were successfully detected. Four highly intense signals of phosphopeptides in human serum were also observed with high signal‐to‐noise ratio and a clear background after enrichment with Ti (IV)@poly(VPA‐co‐EDMA) monolith. It was concluded that the capillary microextraction system enabled fast, efficient and robust enrichment of phosphopeptides from microscale complex samples. The whole enrichment process was completed within 20 min, which was shorter than in the previously reported studies.     

聚多巴胺修饰棉花固定金属离子用于磷酸化多肽的富集

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。     

Highly selective enrichment of phosphopeptides using poly(dibenzo‐18‐crown‐6) as a solid‐phase extraction material

A poly(dibenzo‐18‐crown‐6) was used as a new solid‐phase extraction material for the selective enrichment of phosphopeptides. Isolation of phosphopeptides was achieved based on specific ionic interactions between poly(dibenzo‐18‐crown‐6) and the phosphate group of phosphopeptides. Thus, a method was developed and optimized, including loading, washing and elution steps, for the selective enrichment of phosphopeptides. To assess this potential, tryptic digest of three proteins (α‐ casein, β‐casein and ovalbumin) was applied on poly(dibenzo‐18‐crown‐6). The nonspecific products were removed by centrifugation and washing. The spectrometric analysis was performed using matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry. Highly selective enrichment of both mono‐ and multiphosphorylated peptides was achieved using poly(dibenzo‐18‐crown‐6) as solid‐phase extraction material with minimum interference from nonspecific compounds. Furthermore, evaluation of the efficiency of the poly(dibenzo‐18‐crown‐6) was performed by applying the digest of egg white. Finally, quantum mechanical calculations were performed to calculate the binding energies to predict the affinity between poly(dibenzo‐18‐crown‐6) and various ligands. The newly identified solid‐phase extraction material was found to be a highly efficient tool for phosphopeptide recovery from tryptic digest of proteins.     

Preparation and loading buffer study of polyvinyl alcohol‐based immobilized Ti4+ affinity chromatography for phosphopeptide enrichment

Despite recent advances in phosphoproteomics, an efficient and simple enrichment protocol is still a challenge and of high demand aiming at large‐scale plant phosphoproteomics studies. Here, we developed a novel loading buffer system for synthesized immobilized metal affinity chromatography material targeting plant samples, which was prepared by a simple one‐step esterification between polyvinyl alcohol and phosphoric acid and then was subjected to immobilize Ti⁴⁺. SEM and Fourier transform IR spectroscopy were used to assure the synthesis protocol of the polyvinyl alcohol‐based Ti⁴⁺ immobilized material, and the specific surface areas and pore volumes of the polymers were measured. The selectivity for phosphopeptide enrichment from α‐casein was improved by optimizing the pH and components of the loading buffer. By using potassium hydrogen phthalate/hydrochloric acid with pH at 2.50 as the loading buffer, 19 phosphopeptides with high intensity were identified. The final optimized protocol was adapted to salt‐stressed maize leaves for phosphoproteome analysis. A total of 57 phosphopeptides containing 59 phosphorylated sites from 50 phosphoproteins were identified in salt‐stressed maize leaf. The research was meaningful to obtain much more information about phosphoproteins leading to the comprehension of salt resistance and salt‐inducible phosphorylated processes of maize leaves.     

Profiling Phosphoproteome Landscape in Circulating Extracellular Vesicles from Microliters of Biofluids through Functionally Tunable Paramagnetic Separation

Many biological processes are regulated through dynamic protein phosphorylation. Monitoring disease-relevant phosphorylation events in circulating biofluids is highly appealing but also technically challenging. We introduce here a functionally tunable material and a strategy, extracellular vesicles to phosphoproteins (EVTOP), which achieves one-pot extracellular vesicles (EVs) isolation, extraction, and digestion of EV proteins, and enrichment of phosphopeptides, with only a trace amount of starting biofluids. EVs are efficiently isolated by magnetic beads functionalized with Ti^IV ions and a membrane-penetrating peptide, octa-arginine R₈⁺, which also provides the hydrophilic surface to retain EV proteins during lysis. Subsequent on-bead digestion concurrently converts EVTOP to Ti^IV ion-only surface for efficient enrichment of phosphopeptides for phosphoproteomic analyses. The streamlined, ultra-sensitive platform enabled us to quantify 500 unique EV phosphopeptides with only a few μL of plasma and over 1200 phosphopeptides with 100 μL of cerebrospinal fluid (CSF). We explored its clinical application of monitoring the outcome of chemotherapy of primary central nervous system lymphoma (PCNSL) patients with a small volume of CSF, presenting a powerful tool for broad clinical applications.     

Titanium-containing magnetic mesoporous silica spheres: effective enrichment of peptides and simultaneous separation of nonphosphopeptides and phosphopeptides

Protein phosphorylation is a common posttranslational modification, and involved in many cellular processes. Like endogenous peptides, endogenous phosphopeptides contain many biomarkers of preclinical screening and disease diagnosis. In this work, titanium-containing magnetic mesoporous silica spheres were synthesized and applied for effective enrichment of peptides from both tryptic digests of standard proteins and human serum. Besides, the enriched peptides can be further separated into nonphosphopeptides and phosphopeptides by a simple elution. First, titanium-containing magnetic mesoporous silica spheres were synthesized by a sol-gel method and found to have high surface area, narrow pore size distribution, and useful magnetic responsivity. Then, as the prepared material was used for selective capturing of phosphopeptides, it demonstrated to have higher selectivity than commercial titanium dioxide. Moreover, via combination of size-exclusion mechanism, hydrophobic interaction, and affinity chromatography, titanium-containing magnetic mesoporous silica spheres were successfully applied to simultaneously extract and separate nonphosphopeptides and phosphopeptides from standard protein digestion and human serum.     

Use of polyethylenimine-modified magnetic nanoparticles for highly specific enrichment of phosphopeptides for mass spectrometric analysis

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.     

Use of Sulfonates as Desorption Agents for Phosphopeptide Elution from an Anion-exchange Material

In general, phosphopeptides are specifically adsorbed to the surface of the material at the initial step of phosphopeptide enrichment methods. Thus, nonphosphopeptides can be removed from the media by following the appropriate washing steps. After sufficient washing, the phosphopeptides are eluted from the surface of the material completely for further analysis. Performing the elution of phosphopeptides fully in the enrichment step is very important in terms of determining the whole phosphoproteome profile of a sample by subsequent mass spectrometric analysis. Materials containing anion exchanger groups such as amines on the surface can be used as a selective stationary phase in phosphopeptide enrichment methods. Positively charged groups on the surface of this type of material interact with the phosphate groups of phosphopeptides through electrostatic interactions. Such interactions can be basically manipulated by changing the pH of the medium or replacing the salts present in the solution. Phosphopeptides attached to the surface of anion-exchange materials may be displaced with the addition of highly acidic compounds such as sulfonates to the enrichment medium. Here, we used various sulfonates as desorption agents for the elution of retained phosphopeptides from the surface of an anion-exchange material. We found that differences in the chemical structures and properties of the sulfonates remarkably affected phosphopeptide retrieval from the anion-exchange material.     

Fractionation of phosphopeptides on strong anion‐exchange capillary trap column for large‐scale phosphoproteome analysis of microgram samples

It is one of the key issues to develop powerful fractionating method to increase the identification of the low‐abundance phosphopeptides. In this study, a semi‐online 2‐D LC separation strategy based on three‐step fractionation of the enriched peptides on strong anion‐exchange trap column was developed. It was demonstrated that the sensitivity and phosphoproteome coverage obtained by this fractionating method with strong anion‐exchange trap column is much higher than those by the conventional methods based on C18 trap column. In addition, when the same amount of sample was loaded, the number of identified phosphopeptides had increased 108%. Combination of this three‐step fractionation method with RPLC‐MS/MS analysis by 300 min RP‐gradient separation was applied to phosphoproteome analysis of human liver proteins, and 853 unique phosphopeptides was positively identified from 500 μg tryptic digest of human liver proteins. After three cycles' consecutive analyses, 1554 unique phosphopeptides and 1566 phosphorylated sites were totally identified from 735 phosphorylated proteins at a false discovery rate of <1% in about 54 h of analysis time.     

Fe3O4@Al2O3 magnetic core-shell microspheres for rapid and highly specific capture of phosphopeptides with mass spectrometry analysis

Selective detection of phosphopeptides from complex biological samples is a challenging and highly relevant task in many proteomics applications. In this study, a novel phosphopeptide enrichment approach based on the strong interaction of Fe(3)O(4)@Al(2)O(3) magnetic core-shell microspheres with phosphopeptides has been developed. With a well-defined core-shell structure, the Fe(3)O(4)@Al(2)O(3) magnetic core-shell microspheres not only have a shell of aluminum oxide, giving them a high-trapping capacity for the phosphopeptides, but also have magnetic property that enables easy isolation by positioning an external magnetic field. The prepared Fe(3)O(4)@Al(2)O(3) magnetic core-shell microspheres have been successfully applied to the enrichment of phosphopeptides from the tryptic digest of standard phosphoproteins beta-casein and ovalbumin. The excellent selectivity of this approach was demonstrated by analyzing phosphopeptides in the digest mixture of beta-casein and bovine serum albumin with molar ratio of 1:50 as well as tryptic digest product of casein and five protein mixtures. The results also proved a stronger selective ability of Fe(3)O(4)@Al(2)O(3) magnetic core-shell microspheres over Fe(3+)-immobilized magnetic silica microspheres, commercial Fe(3+)-IMAC (immobilized metal affinity chromatography) resin, and TiO(2) beads. Finally, the Al(2)O(3) coated Fe(3)O(4) microspheres were successfully utilized for enrichment of phosphopeptides from digestion products of rat liver extract. These results show that Fe(3)O(4)@Al(2)O(3) magnetic core-shell microspheres are very good materials for rapid and selective separation and enrichment of phosphopeptides.     

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。     

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.     

Two‐step Immobilized Metal Affinity Chromatography (IMAC) for Phosphoproteomics Using Mass Spectrometry

In this study, a new strategy named two‐step IMAC is demonstrated as a novel prelude to MS analysis of phosphoproteome by increasing the enrichment factor of phosphoproteins/phosphopeptides from a protein mixture. In this method, the first IMAC was performed at the protein level to extract the minute amount of phosphoproteins present in the sample. During this step, nonphosphoproteins and other undesired chemicals or inhibitors were excluded. After tryptic digestion, the second IMAC was performed at the peptide level to enrich phosphopeptides present in the tryptic digest, and the eluent from the second IMAC was analyzed by MALDI‐MS. It is particularly noticeable that the eluent from the first IMAC can be directly digested by trypsin without buffer exchange. Our results revealed that β‐casein that was spiked in a protein mixture can be successfully extracted by the first IMAC at a concentration of less than 1–3%, and the two phosphopeptides of β‐casein with single and four phosphorylation sites, respectively, can be captured by the second IMAC. It was found that the two‐step IMAC method could significantly reduce non‐specific bindings from unwanted proteins and greatly enhance the MALDI‐MS signal of phosphopeptide ions compared to the typical one‐step IMAC, by which only IMAC at the peptide level was performed. Two‐step IMAC was also found to tolerate a greater amount and a greater concentration range of proteins than one‐step IMAC, which is especially important when analyzing complicated unknown samples. Furthermore, the MS signal of phosphopeptide ions did not appear to be degraded by the presence of biological matrixes, such as the cell lysate in which the β‐casein was spiked in.     

Sequential Fe3O4/TiO2 enrichment for phosphopeptide analysis by liquid chromatography/tandem mass spectrometry

Protein phosphorylation regulates a wide range of cellular functions and is associated with signaling pathways in cells. Various strategies for enrichment of phosphoproteins or phosphopeptides have been developed. Here, we developed a novel sequential phosphopeptide enrichment method, using magnetic iron oxide (Fe₃O₄) and titanium dioxide (TiO₂) particles, to detect mono‐ and multi‐phosphorylated peptides. In the first step, phosphopeptides were captured on Fe₃O₄ particles. In a subsequent step, any residual phosphopeptides were captured on TiO₂ particles. The particles were eluted and rinsed to yield phosphopeptide‐enriched fractions that were combined and analyzed using liquid chromatography/tandem mass spectrometry (LC/MS/MS). The validity of this sequential Fe₃O₄/TiO₂ enrichment strategy was demonstrated by the successful enrichment of bovine α‐casein phosphopeptides. We then applied the sequential Fe₃O₄/TiO₂ enrichment method to the analysis of phosphopeptides in L6 muscle cell lysates and successfully identified mono‐ and multi‐phosphorylated peptides. Copyright © 2010 John Wiley & Sons, Ltd.     

In‐situ enrichment of phosphopeptides on MALDI plates modified by ambient ion landing

We report substantial in‐situ enrichment of phosphopeptides in peptide mixtures using titanium and zirconium dioxide‐coated matrix assisted laser desorption‐ionization (MALDI) plates prepared by recently reported ambient ion landing deposition technique. The technique was able to modify four common materials currently used for MALDI targets (stainless steel, aluminum, indium‐tin oxide glass and polymeric anchor chip). The structure of the deposited dioxide was investigated by electron microscopy, and different surfaces were compared and discussed in this study. Two standard proteins were used to test the enrichment capabilities of modified MALDI plates: casein and in‐vitro phosphorylated trehalase. The enrichment of casein tryptic digest resulted in identification of 20 phosphopeptides (including miscleavages). Trehalase was used as a suitable model of larger protein that provided more complex peptide mixture after the trypsin digestion. All four possible phosphorylation sites in trehalase were identified and up to seven phosphopetides were found (including methionine oxidations and miscleavages). Two different mass spectrometers, MALDI‐Fourier transform ion cyclotron resonance (FTICR) and MALDI‐time of flight, were used to detect the phosphopeptides from modified MALDI plates after the enrichment procedure. It was observed that the desorption‐ionization phenomena on the modified surfaces are not critically influenced by the parameters of the different MALDI ion sources (e.g. different pressure, different extraction voltages), and thus the presence of dioxide layer on the standard MALDI plate does not significantly interfere with the main MALDI processes. The detection of phosphopeptides after the enrichment could be done by both instruments. Desorption electrospray ionization coupled to the FTICR was also tested, but, unlike MALDI, it did not provide satisfactory results. Copyright © 2012 John Wiley & Sons, Ltd.     

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.     

基于强阳离子交换色谱分离的磷酸化肽段富集策略

为了在短时间内获得相对含量高的磷酸化肽段,以标准磷酸化蛋白质为模型对强阳离子交换色谱（SCX）分离磷酸化肽段体系的缓冲溶液和梯度设置进行了研究,并用酵母酶切肽段混合物考察了该路线在较复杂的样品中的应用。实验结果表明优化后的体系能够在30 min内分离出磷酸肽段,而且非磷酸化肽段的干扰很少,这样便相对提高了磷酸化肽段在质谱仪中的响应强度,重要的是该体系可以对复杂样品进行很好的分离。这说明SCX用于规模化磷酸化肽段富集的策略是可行的。本研究为磷酸化蛋白质组学规模化分析提供了实用技术。     

基于亲和色谱的肺癌细胞磷酸化蛋白质组研究及其应用

磷酸化是蛋白质翻译后修饰的重要形式之一,其异常往往会导致细胞内信号通路的紊乱和疾病的发生。固定化金属离子亲和色谱(IMAC)是磷酸化肽段的高效富集技术,在磷酸化蛋白质组研究方面应用广泛。该研究以金属钛离子(Ti⁴⁺)螯合IMAC材料(Ti⁴⁺-IMAC)为载体,进行磷酸化肽段富集。比较了10 μm Ti⁴⁺-IMAC通过振荡法和固相萃取法(SPE)富集磷酸肽的效果,发现振荡法可以富集到更多的磷酸肽;对比了两种尺寸(10 μm和30 μm)Ti⁴⁺-IMAC在磷酸化肽段富集中的差异,发现小尺寸材料富集效果更佳。进一步采用优化的策略比较了不同转移能力肺癌细胞的磷酸化蛋白质组,免标记定量蛋白质组学结果表明,优化的Ti⁴⁺-IMAC方法可以从正常的肺成纤维细胞MRC5、低转移肺癌细胞95C和高转移肺癌细胞95D中分别鉴定到510、863和1108种磷酸化蛋白质,其中317种为3组所共有。该研究共鉴定到1268种磷酸化蛋白质上的7560个磷酸化位点,其中1130个为差异磷酸化位点,文献报道显示部分异常表达的激酶与癌症转移密切相关。通过生信对比分析发现,异常表达的磷酸化蛋白质主要与细胞侵袭、迁移和死亡等细胞迁移方面的功能有关。通过优化磷酸化肽富集策略,初步阐明了磷酸化蛋白质网络的异常与肺癌转移之间的相关性,该方法有望用于肺癌进展相关的磷酸化位点、磷酸化蛋白质及其信号通路研究。     

精氨酸功能化磁性纳米材料的制备及在磷酸化肽富集中的应用

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.