Phosphoprotein Analysis by MALDI-TOF Mass Spectrometry using On-Probe Tandem Proteolysis and Dephosphorylation

In this investigation, methods based on on-probe enzymatic cleavage matrix-assisted laser desorption/ionization time-of-flight mass spectrometric (MALDI-TOF-MS) analyses have been developed, allowing the rapid assignment of phosphorylation sites within phosphoproteins. The procedures involved robotic sample deposition of a phosphoprotein, such as intact bovine β-casein, on stainless steel or gold MALDI plates, on-probe proteolysis with trypsin for 10–180?s at 37°C, on-probe dephosphorylation for 1–10?min at 37°C with alkaline phosphatase, followed by differential mass spectrometry with peptide mass mapping. The dephosphorylation conditions were initially optimized using in-solution tryptic digestion of the phosphoprotein performed in the presence of MS-compatible anionic surfactant sodium 3-[(2-methyl-2-undecyl-1,3-dioxolan-4-yl)methoxy]-1-propanesulfonate. Two methods of trypsin deactivation were investigated, cooling and quenching by acidification, which resulted in the surfactant either staying intact or becoming cleaved, respectively. Since the surfactant had no detrimental effects on dephosphorylation of phosphopeptides, the acidification and neutralization steps were not included in the final analytical method. A protocol, comprising on-probe tandem, surfactant-aided proteolysis for 3?min followed by on-probe dephosphorylation for 10?min was thus established, allowing the rapid identification of location and sequence of phosphopeptides within a phosphoprotein by these procedures.     

Preparation of mesoporous carbon from commercial activated carbon with steam activation in the presence of cerium oxide

Mesoporous carbon was prepared from the commercial activated carbon by steam activation with cerium oxide as catalyst. Steam activation with a catalyst loading of 0.5-2.0 wt% at 680-870 degrees C was examined. The surface area and pore size were evaluated by nitrogen adsorption at 77 K, and the structure of cerium oxide was characterized by XRD, XPS, and TEM. The results showed that the catalyst promoted the development of a mesopore at lower temperature (680-740 degrees C), and the mesopore was concentrated around 4-10 nm. The noncatalytic activation was advantageous in mesopore development and the catalyst would restrict the formation of mesopores at high temperature (800-870 degrees C). Higher loading of cerium oxide and higher activation temperature caused the aggregation of cerium oxide and then resulted in scattered pore size distribution.     

Efficient enrichment and identification of phosphopeptides by cerium oxide using on-plate matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis

An efficient and simple method for enrichment and identification of phosphopeptides by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) using cerium oxide is presented. After pretreatment of tryptic digests of phosphoproteins with CeO(2), nonphosphopeptides are discarded and phosphopeptides are enriched. By applying the separated CeO(2) on a target plate and analysis using MALDI-TOF MS, peaks of phosphopeptides and their correspondingly series of dephosphorylated peptides are observed in the mass spectra. Thus, the phosphopeptides are very easy to identify with the mass difference, which are all 80 Da between adjacent peaks in the same series, and clear background in the spectra owing to elimination of signal suppression from large amounts of nonphosphopeptides. Furthermore, the phosphopeptides can be dephosphorylated completely after a further NH(4)OH elution. Tryptic digest products from several standard proteins are pretreated using CeO(2) to demonstrate the efficiency of this method. Phosphopeptides from a very small quantity of human serum are enriched and analyzed, and proteins also identified by searching against a database using Mascot on MALDI-TOF/TOF fragments, which indicates that this method may be employed in complex samples for further application.     

Preparation and characterization of ceria‐zirconia composite for enrichment and identification of phosphopeptides

Ceria‐zirconia composites at different molar ratios were synthesized. Several methods were used to characterize these composites, including X‐ray photoelectron spectroscopy, surface area and surface acid‐base property detection. A one‐step method for isolation and identification of phosphopeptides from peptide mixture was created using these ceria‐zirconia composites. Using tryptic digest of standard phosphorylated protein, we have shown that these enrichment and dephosphorylation activities are effective. The adsorption capacity and catalytic property of ceria‐zirconia composites at different molar ratios and calcinated temperatures were studied. In combination with MALDI‐TOF‐based peptide mass finger printing technique, we have established a method to utilize the enrichment/dephosphorylation dual properties of these ceria‐zirconia composites for the analysis of phosphoprotein in nonfat milk successfully.     

Solid-state synthesis of monazite-type compounds containing tetravalent elements

On the basis of optimized grinding/heating cycles developed for several phosphate-based ceramics, the preparation of brabantite and then monazite/brabantite solid solutions loaded with tetravalent thorium, uranium, and cerium (as a plutonium surrogate) was examined versus the heating temperature. The chemical reactions and transformations occurring when heating the initial mixtures of AnO2/CeO2, CaHPO(4).2H2O (or CaO), and NH4H2PO4 were identified through X-ray diffraction (XRD) and thermogravimetric/differential thermal analysis experiments. The incorporation of thorium, which presents only one stabilized oxidation state, occurs at 1100 degrees C. At this temperature, all the thorium-brabantite samples appear to be pure and single phase as suggested by XRD, electron probe microanalyses, and micro-Raman spectroscopy. By the same method, tetravalent uranium can be also stabilized in uranium-brabantite, i.e., Ca0.5U0.5PO4, after heating at 1200 degrees C. Both brabantites, Ca0.5Th0.5PO4 and Ca0.5U0.5PO4, begin to decompose when increasing the temperature to 1400 and 1300 degrees C, respectively, leading to a mixture of CaO and AnO2 by the volatilization of P4O10. In contrast to the cases of thorium and uranium, cerium(IV) is not stabilized during the heating treatment at high temperature. Indeed, the formation of Ca0.5Ce0.5PO4 appears impossible, due to the partial reduction of cerium(IV) into cerium(III) above 840 degrees C. Consequently, the systems always appear polyphase, with compositions of CeIII1-2xCeIVxCaxPO4 and Ca2P2O7. The same conclusion can be also given when discussing the incorporation of cerium(IV) into La1-2xCeIIIx-yCeIVyCay(PO4)1-x+y. This incomplete incorporation of cerium(IV) confirms the results obtained when trying to stabilize tetravalent plutonium in Ca0.5PuIV0.5PO4 samples.     

Magnetic iron oxide nanoparticle enrichment of phosphopeptides on a radiate microstructure MALDI chip

Several methods can be used to improve the enrichment of phosphorylated proteins. In this paper, phosphopeptides were enriched using magnetic iron(II,III) oxide (magnetite, Fe(3)O(4)) nanoparticles (NPs) on a radiate microstructure silicon chip and then analyzed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) without further purification processes. We have developed a radiate microstructure chip on which samples can be concentrated for analysis by MALDI-TOFMS. The phosphoprotein digests and magnetic iron oxide NPs aqueous solution were deposited onto the central zone of the radiate microstructure silicon chip and enabled the on-chip enrichment of phosphopeptides. Microscopic analysis confirmed that the applied samples were confined to the central zone. Sample spots focused on the chip were much smaller than those on an unmodified plate with the same total volume. Different additives were used and optimized processes were performed to minimize non-phosphopeptides interference. These data collectively demonstrate that our on-chip phosphopeptide enrichment protocol is a rapid and easy-to-use method for phosphoproteome analysis.     

Chemical dephosphorylation for identification of multiply phosphorylated peptides and phosphorylation site determination

We have developed a novel strategy to improve the efficiency of identification of multiply phosphorylated peptides isolated by hydroxy acid modified metal oxide chromatography (HAMMOC). This strategy consists of alkali‐induced chemical dephosphorylation (beta‐elimination reaction) of phosphopeptides isolated by HAMMOC prior to analysis by liquid chromatography/mass spectrometry (LC/MS). This approach identified 1.9‐fold more multiply phosphorylated peptides than the conventional approach without beta‐elimination from a digested mixture of three standard phosphoproteins. In addition, the accuracy of phosphorylation site determination in synthetic phosphopeptides was significantly improved. Finally, we applied this approach to a cell lysate. By combining this dephosphorylation approach with the conventional approach, we successfully identified 1649 unique phosphopeptides, including 325 multiply phosphorylated phosphopeptides, from 200 µg of cultured Arabidopsis cells. These results indicate that chemical dephosphorylation prior to LC/MS analysis increases the efficiency of identification of multiply phosphorylated peptides, as well as the accuracy of phosphorylation site determination. Copyright © 2010 John Wiley & Sons, Ltd.     

Site‐specific separation and detection of phosphopeptide isomers with pH‐mediated stacking capillary electrophoresis‐electrospray ionization‐tandem mass spectrometry

This study reported a pH‐mediated stacking CE coupled with ESI MS/MS method to determine the phosphorylation sites of three synthetic phosphopeptides containing structural isomers. These phosphopeptides mimic the phosphopeptides (amino acid residues 12–25) derived from the trypsin‐digested products of human lamin A/C protein. The LODs were determined to be 118, 132 and 1240 fmol for SGAQASS¹⁹TpPL²²SPTR, SGAQASS¹⁹TPL²²SpPTR, and SGAQASS¹⁹TpPL²²SpPTR, respectively. The established method was employed to analyze the phosphorylation sites of the trypsin‐digested products of glutathione S‐transferase‐lamin A/C (1–57) fusion protein that had been phosphorylated in vitro by cyclin‐dependent kinase 1. The results indicated that this method is feasible to specifically determine the phosphorylation site from phosphopeptide isomers in the trypsin‐digested products of a kinase‐catalyzed phosphoprotein, which should benefit the investigation of protein kinase‐mediated cellular signal transduction.     

Synthesis and Characterization of Indium-borate Glass-ceramics Containing Ho0.01Ce0.74Zr0.25O1.995 Nanorods via Incorporation Method

Glasses in the system 5In₂O₃·94Na₂B₄O₇ were fabricated via melt quenching technique. The amorphous nature of the quenched glasses was confirmed by X‐ray powder diffraction studies, and the infrared spectra of the glasses show no boroxol ring formation in the structure of these glasses. Differential thermal analysis is shown glass transition temperature 696°C and crystallization temperature 1151°C. A cerium‐zirconium mixed oxide Ce_0.75Zr_0.25O₂ and Ho‐doped cerium‐zirconium mixed oxide were obtained by solid‐state method. Then glass powder and Ho‐doped cerium‐zirconium mixed oxide were mixed. The mixture was heated in a crucible. The glass‐ceramic sample was obtained by pouring the melts on stainless steel. Obtained samples were annealed at 450°C for 1 h to remove thermal strain. Differential thermal analysis for glass‐ceramic sample is shown glass transition temperature 668°C and crystallization temperature 1159°C. The scanning electron microscopy study for glass‐ceramic indicates that the crystallized glass consists of rod‐like crystals with average diameter of about 38 nm dispersed in the glassy regions.     

Synthesis and characterization of volatile,fluorine-free beta-ketoiminate lanthanide MOCVD precursors and their implementation in low-temperature growth of epitaxial CeO(2) buffer layers for superconducting electronics

A new class of volatile, low-melting, fluorine-free lanthanide metal-organic chemical vapor deposition (MOCVD) precursors has been developed. The neutral, monomeric Ce, Nd, Gd, and Er complexes are coordinatively saturated by a versatile, multidentate ether-functionalized beta-ketoiminato ligand series, the melting point and volatility characteristics of which can be tuned by altering the alkyl substituents on the keto, imino, and ether sites of the ligand. Direct comparison with conventional lanthanide beta-diketonate complexes reveals that the present precursor class is a superior choice for lanthanide oxide MOCVD. Epitaxial CeO(2) buffer layer films can be grown on (001) YSZ substrates by MOCVD at significantly lower temperatures (450-650 degrees C) than previously possible by using one of the newly developed cerium beta-ketoiminate precursors. Films deposited at 540 degrees C have good out-of-plane (Deltaomega = 0.85 degrees ) and in-plane (Deltaphi = 1.65 degrees ) alignment and smooth surfaces (rms roughness approximately 4.3 A). The film growth rate decreases and the films tend to be smoother as the deposition temperature is increased. High-quality yttrium barium copper oxide (YBCO) films grown on these CeO(2) buffer layers by pulsed organometallic molecular beam epitaxy exhibit very good electrical transport properties (T(c) = 86.5 K, J(c) = 1.08 x 10(6) A/cm(2) at 77.4 K).     

Development of an integrated chromatographic system for on-line digestion and characterization of phosphorylated proteins

The development of an integrated chromatographic system for complete phosphoprotein analysis is described. The digestion of phosphoproteins with trypsin- or pronase-based monolithic bioreactors is carried out on-line with selective enrichment on a TiO(2) trap and separation of the produced phosphopeptides by reversed-phase liquid chromatography-multiple mass spectrometry (RPLC/MS(n)). A detailed study on the selective extraction of peptides with different degrees of phosphorylation on TiO(2) cartridges is discussed. This analytical strategy has been optimized using beta-casein as a standard phosphoprotein, and then applied to the identification of phosphorylation sites in insulin-like grow factor-binding protein 1 (IGFBP-1) isolated from amniotic fluid.     

Oxygen exchange properties in the new pyrochlore solid solution Ce2Sn2O7-Ce2Sn2O8

The phase-pure cerium stannate pyrochlore (Ce₂Sn₂O₇) has been prepared for the first time. The structure and oxidation states of both cations were carefully reviewed, and the compound was unambiguously replaced within the rare-earth stannate series. As a consequence of the low stability of trivalent cerium in oxide phases, one oxygen per formula unit could be intercalated by calcination under O₂ at 400 °C, leading to the new Ce₂Sn₂O₈ pyrochlore. This latter phase is subject to oxygen under-stoichiometry from 400 to 700 °C. However, oxygen deintercalation seems to be in competition with cerium oxide segregation at high temperature, leading to the formation of cerium deficient pyrochlore phases.     

Surfactant mediated synthesis of spherical binary oxides photocatalytic with enhanced activity in visible light

Spherical silica and zirconia mixed titania and pure titania samples were prepared in presence of cetyltrimethylammonium bromide (CTAB) through controlled hydrolysis of corresponding metal alcoxides. Effect of surfactant amount and calcinations temperature on morphology, surface area and photocatalytic activity is studied using PXRD, SEM, FTIR, Solid state UV-vis spectroscopy and BET surface area. It is well observed that in presence of 2 mol% CTAB, uniform sized spherical oxide particles can be synthesized. However, increasing or decreasing the surfactant amount does not favor the spherical particle formation. Material synthesis in presence of CTAB not only helps in the spherical particle formation but also increases the surface area and visible light absorption. Studies on photocatalytic lead removal with respect to calcination temperature indicate that the calcination at 500 degrees C is most suitable for the best photocatalytic activity. Mixing of zirconia and silica helps in anatase phase stabilization even at 900 degrees C calcination. Accordingly low decrease in surface area even at 900 degrees C calcination is observed. Due to the phase stabilization and higher surface area binary oxide materials showed comparatively better photocatalytic activity even after calcination at 900 degrees C. So it can be concluded that present synthesis approach can produce uniform sized spherical binary oxide materials with better photocatalytic activity in visible light.     

酪氨酸磷酸化多肽的化学选择性富集

酪氨酸激酶在生物分子的信号转导中起着非常重要的作用,目前除抗体技术外尚无有效的化学方法能够实现对酪氨酸磷酸化蛋白或多肽的选择性富集,然而抗体通常成本较高,而且往往会有模体序列的选择性识别。本文发展了一种基于化学反应的酪氨酸磷酸化肽段的选择性富集,该方法利用了β消除反应只能发生在丝氨酸和苏氨酸磷酸化多肽的特性,以反相选择方法,从而实现对酪氨酸磷酸化肽段的选择性富集。以标准多肽对其反应效率和回收率进行了考察,20分钟内丝氨酸磷酸化多肽的β消除反应效率可达99%以上,而同时酪氨酸磷酸化肽段可保持70%的回收率。进一步以六种标准蛋白混合物的酶解产物对其进行考察,经β消除反应和亲和富集之后,只有酪氨酸磷酸化多肽可以被检测出来,该方法为蛋白质酪氨酸磷酸化的分析提供了一种新的手段。     

Correlation of the oxidation state of cerium in sol-gel glasses as a function of thermal treatment via optical spectroscopy and XANES studies

Sol-gel glass matrices containing lanthanides have numerous technological applications and their formation involves several chemical facets. In the case of cerium, its ability to exist in two different oxidation states or in mixed valence state provides additional complexities for the sol-gel process. The oxidation state of cerium present during different facets of preparation of sol-gel glasses, and also as a function of the starting oxidation state of cerium added, were studied both by optical spectroscopy and X-ray absorption near-edge structures (XANES). The findings acquired by each approach were compared. The primary focus was on the redox chemistries associated with sample preparation, gelation, and thermal treatment. When Ce3+ is introduced into the starting sols, the trivalent state normally prevails in the wet and room temperature-dried gels. Heating in air at >100 degrees C can generate a light yellow coloration with partial oxidation to the tetravalent state. Above 200 degrees C and up to approximately 1000 degrees C, cerium is oxidized to its tetravalent state. In contrast, when tetravalent cerium is introduced into the sol, both the wet and room temperature-dried gels lose the yellow-brown color of the initial ceric ammonium nitrate solution. When the sol-gel is heated to 110 degrees C it turns yellowish as the cerium tends to be re-oxidized. The yellow color is believed to represent the effect of oxidation and oligomerization of the cerium-silanol units in the matrix. The luminescence properties are also affected by these changes, the details of which are reported herein.     

Cerium oxide nanoparticle-mediated self-assembly of hybrid supramolecular hydrogels

Hybrid supramolecular hydrogels are prepared by non-enzymatic dephosphorylation of N-fluorenylmethyloxycarbonyl tyrosine-(O)-phosphate (FMOC-Tyr-P) using catalytic cerium oxide nanoparticles. The organic-inorganic hydrogels exhibit enhanced viscoelastic properties compared with analogous materials prepared using alkaline phosphatase.     

Coupling of TiO(2)-mediated enrichment and on-bead guanidinoethanethiol labeling for effective phosphopeptide analysis by matrix-assisted laser desorption/ionization mass spectrometry

Titanium dioxide (TiO2)-mediated phosphopeptide enrichment has been introduced as an effective method for extracting phosphopeptides from highly complex peptide mixtures. Chemical labeling by beta-elimination/Michael addition is also useful for increasing mass intensity in phosphopeptide analysis. Both of these methods were coupled in order to simultaneously enrich phosphopeptides and allow for detection and sequencing of the enriched peptides with high mass sensitivity. Phosphopeptides were successfully enriched on TiO2 beads without the use of any hydroxy acid additives like 2,5-dihydroxybenzoic acid. Labeling was accomplished on-bead with a guanidinoethanethiol (GET) tag containing a guanidine moiety. These GET-labeled derivatives were detected by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). GET labeling converted phosphoserine into guanidinoethylcysteine, a structural arginine-mimic. In particular, GET-labeled lysine-terminated phosphopeptides showed dramatically increased peak intensities compared to those of the corresponding intact phosphopeptides. Additionally, the on-bead labeling minimized manipulation steps and sample loss. The coupled technique was also further validated by applying to the analysis of phosphopeptides from complex tryptic digests of phosphoprotein mixtures.     

Synthesis and Photocatalytic Activity of Cerium(IV) Fibrous Nanostructures

The method using cellulose as a template was proposed for the synthesis of fibrous structures consisting of cerium(IV) oxide nano-sized particles. The chemical composition and morphology of the samples as functions of the annealing temperature were studied by X-ray diffraction analysis, IR spectroscopy, and scanning electron microscopy. It was shown that CeO2 particles obtained from Ce(NO3)3 by the template synthesis at 800°C have the highest photocatalytic activity in the photodestruction of methyl orange (pH = 3.5, acetate buffer) and phenol. It was revealed that CeO2 particles exhibit photoprotective properties in the hydrotartrate buffer solution of methyl orange.     

基于碳纳米管的氧化铈纳米管的合成及表征

建立了一种制备氧化铈纳米管的新方法, 即以碳纳米管(CNTs)为模板, 在常温常压下采用液相沉积法在CNTs表面包覆CeO2, 通过煅烧除去CNTs模板, 得到氧化铈纳米管. 通过透射电子显微镜(TEM)、X射线衍射(XRD)和X射线光电子能谱(XPS)对其形貌和结构进行了表征. 所得CeO2纳米管为面心立方结构, 直径40~60 nm, 长度0.5~2 μm.     

基于MALDI-TOF MS的串联富集策略在磷酸化蛋白组学中的应用

提出一种除盐-富集串联用于磷酸肽富集研究的思路。选用C18柱和铈(Ⅳ)修饰的壳聚糖材料进行脱盐实验,以制备的基于聚合物基体螯合Fe3+的亲和色谱材料为富集材料。将直接富集和串联策略应用到标准品和血清中,研究结果表明,该富集材料具有高选择性和高灵敏度(1.6 fmol),铈(Ⅳ)修饰的壳聚糖材料前提下的串联策略能明显降低样品的复杂性。相比直接富集方法,能够提高磷酸化肽的覆盖率。