Software for quantitative proteomic analysis using stable isotope labeling and data independent acquisition

Many software tools have been developed for analyzing stable isotope labeling (SIL)-based quantitative proteomic data using data dependent acquisition (DDA). However, programs for analyzing SIL-based quantitative proteomics data obtained with data independent acquisition (DIA) have yet to be reported. Here, we demonstrated the development of a new software for analyzing SIL data using the DIA method. Performance of the DIA on SYNAPT G2MS was evaluated using SIL-labeled complex proteome mixtures with known heavy/light ratios (H/L = 1:1, 1:5, and 1:10) and compared with the DDA on linear ion trap (LTQ)-Orbitrap MS. The DIA displays relatively high quantitation accuracy for peptides cross all intensity regions, while the DDA shows an intensity dependent distribution of H/L ratios. For the three proteome mixtures, the number of detected SIL-peptide pairs and dynamic range of protein intensities using DIA drop stepwise, whereas no significant changes in these aspects using DDA were observed. The new software was applied to investigate the proteome difference between mouse embryonic fibroblasts (MEFs) and MEF-derived induced pluripotent stem cells (iPSCs) using (16)O/(18)O labeling. Our study expanded the capacities of our UNiquant software pipeline and provided valuable insight into the performance of the two cutting-edge MS platforms for SIL-based quantitative proteomic analysis today.     

Ionic conductivity of electrolytes formed from PEO-LiCF3SO3 complex low molecular weight poly(ethylene glycol)

Films of poly(ethylene oxide)-LiCF₃SO₃-based complexes containing different amounts of poly(ethylone glycol) (PEG) with molecular weights ranging from 400 to 2000 were prepared by solution casting. The ionic conductivity is presented as a function of temperature, molecular weight and the PEG content used. The conductivity increases with decreasing molecular weight of PEG and with increasing PEG content. The incorporation of PEG with a molecular weight of 600 or less gives rise to a maximum conductivity value of 3 × 10^–3 Sm^–1 at 25° C. The conductivity enhancement at room temperature can be attributed to the increase in the amorphous regions responsible for the ionic conduction.     

Electron-beam-initiated polymerization of poly(ethylene glycol)-based wood impregnants

The current study demonstrates that methacrylate and acrylate poly(ethylene glycol) (PEG) functional oligomers can be effectively impregnated into wood blocks, and cured efficiently to high conversions without catalyst by e-beam radiation, allowing for less susceptibility to leaching, and favorable properties including higher Brinell hardness values. PEG based monomers were chosen because there is a long history of this water-soluble monomer being able to penetrate the cell wall, thus bulking it and decreasing the uptake of water which further protects the wood from fungal attack. Diacrylate, dimethacrylate, and dihydroxyl functional PEG of M(w) 550-575, of concentrations 0, 30, 60, and 100 wt % in water, were vacuum pressure impregnated into Scots Pine blocks of 15 × 25 × 50 mm in an effort to bulk the cell wall. The samples were then irradiated and compared with nonirradiated samples. It was shown by IR, DSC that the acrylate polymers were fully cured to much higher conversions than can be reached with conventional methods. Leaching studies indicated a much lower amount of oligomer loss from the cured vinyl functional PEG chains in comparison to hydroxyl functional PEG indicating a high degree of fastening of the polymer in the wood. The Brinell hardness indicated a significant increase in hardness to hardwood levels in the modified samples compared to the samples of hydroxyl functional PEG and uncured vinyl PEG samples, which actually became softer than the untreated Scots Pine. By monitoring the dimensions of the sample it was found by weight percent gain calculations (WPG %) that water helps to swell the wood structure and allow better access of the oligomers into the cell wall. Further, the cure shrinkage of the wood samples demonstrated infiltration of the oligomers into the cell wall as this was not observed for methyl methacrylate which is well-documented to remain in the lumen. However, dimensional stability of the vinyl polymer modified blocks when placed in water was not observed to the same extent as PEG.     

Poly(ethylene glycol) decorated poly(methylmethacrylate) nanoparticles for protein adsorption

Poly(ethylene glycol) decorated poly(methyl methacrylate) particles were synthesized by means of emulsion polymerization using poly(ethylene glycol) sorbitan monolaurate (Tween-20) as surfactant. PMMA/PEG particles presented mean diameter (195 ± 15) nm, indicating narrow size distribution. The adsorption behavior of bovine serum albumin (BSA) and concanavalin A (ConA) onto PMMA/PEG particles was investigated by means of spectrophotometry. Adsorption isotherms obtained for BSA onto PMMA/PEG particles fitted well sigmoidal function, which is typical for multilayer adsorption. Con A adsorbed irreversibly onto PMMA/PEG particles. The efficiency of ConA covered particles to induce dengue virus quick agglutination was evaluated.     

Poly(vinyl pyridine-poly ethylene glycol methacrylate-ethylene glycol dimethacrylate) beads for heavy metal removal

Poly(vinyl pyridine-poly ethylene glycol methacrylate-ethylene glycol dimethacrylate) [poly(VP-PEGMA-EGDMA)] beads with an average size of 30–100 μm were prepared by suspension polymerization. Poly(VP-PEGMA-EGDMA) beads were characterized by swelling studies, scanning electron microscopy (SEM), elemental analysis, Fourier Transform Infrared Spectroscopy (FTIR). The beads with a swelling ratio of 65% were used for the heavy metal removal studies. Chelation capacity of the beads for the selected metal ions, i.e., Pb(II), Cd(II), Cr(III) and Cu(II) were investigated in aqueous media containing different amounts of these ions (5–80 mg/l) and at different pH values (2.0–10.0). The maximum chelation capacities of the poly(VP-PEGMA-EGDMA) beads were 18.23 mg/g for Pb(II), 16.50 mg/g for Cd(II), 17.38 mg/g for Cr(III) and 18.25 mg/g for Cu(II). The affinity order on mass basis was observed as follows: Cu(II) > Pb(II) > Cr(III) > Cd(II). pH significantly affected the chelation capacity of VP incorporated beads. Heavy metal adsorption on the poly(PEGMA-EGDMA) control microspheres was negligible. Regeneration of the chelating beads was easily performed with 0.1 M HNO₃. It was shown that these beads can be used effectively for heavy metal removal from aqueous solutions with repeatedly adsorption–desorption operations. These features show that poly(VP-PEGMA-EGDMA) beads are potential candidate sorbent for heavy metal removal.     

Poly(ethylene glycol)-functionalized devices for electric field gradient focusing

Electric field gradient focusing (EFGF) is an equilibrium gradient focusing technique that depends on an electric field gradient and a hydrodynamic counterflow to focus, concentrate, and separate charged analytes. In this work, EFGF devices were fabricated from poly(ethylene glycol) (PEG)-functionalized acrylic plastic. The separation channel was formed in an ionically conductive and protein-resistant PEG-functionalized hydrogel, which was cast in a changing cross-sectional cavity in the plastic device. A linear electric field gradient was obtained by applying a voltage lengthwise across the shaped hydrogel. Standard proteins were used as analytes to demonstrate the performance of these EFGF devices. With an increase in counterflow rate or decrease in applied voltage, analyte bands broadened, but resolution increased in agreement with theory. To reduce analyte band dispersion and improve focusing performance, a protein-compatible PEG-functionalized monolith was incorporated in the EFGF channel. Compared with focusing in an open channel, protein bands in the monolith-filled EFGF channel were significantly narrower.     

Poly(ethylene glycol) enhanced dehydrochlorination of poly(vinyl chloride)

Poly(ethylene glycol) (PEG), an environment-friendly reaction medium, has been adopted to accelerate the dehydrochlorination of poly(vinyl chloride) (PVC). Experimental results demonstrated that at 210 degrees C for 1h the dechlorination degree was as high as 74.2% for PVC/PEG, while for PVC only 50.0%. Moreover, from thermogravimetric analysis, it was found that for PVC/PEG the decomposition of PVC corresponding to the dehydrochlorination stage shifted to lower temperatures compared with that of pure PVC, suggesting some interactions exist between PEG and PVC that caused the faster dehydrochlorination rate. In addition, during this process, no waste byproducts such as KCl have been produced, and satisfactory recyclability of PEG (10 cycles) has been obtained.     

熔融缩聚合成聚乳酸-聚乙二醇共聚物及其性能研究

采用羧基封端乳酸预聚物与聚乙二醇熔融缩聚合成了聚乳酸-聚乙二醇共聚物,并用GPC、FTIR、1H-NMR等方法表征了预聚物与共聚物,结果表明,预聚物的羧基封端率高于95%,预聚物的相对分子质量可由投料比(物质的量比)控制.热分析结果表明,共聚物中聚乳酸链段呈无规分布,而聚乙二醇链段能够形成结晶微区.力学性能测试结果表明,共聚物的断裂伸长率达371%,有望在聚乳酸韧性改性方面得到应用.     

Impact compressibility of a poly(ethylene glycol)-based nanocomposite fluid

The behavior of a poly(ethylene glycol)-based nanocomposite shear thickening fluid (STF) under impact loading conditions has been experimentally studied using the Kolsky method and related techniques. The dependence of the pressure in the STF on the volume strain magnitude has been determined. It is established that the radial and axial components of the stress tensor almost coincide, which shows that the material behaves like an incompressible liquid. The character of the stress-strain curves (hysteresis) indicates that the STF is characterized by some energy dissipation in the load-unload cycle.     

不同相对分子质量聚乙二醇增塑聚乳酸共混物的制备与性能

文中探究不同相对分子质量聚乙二醇(PEG)对聚乳酸(PLA)增塑改性的影响。采用转矩流变仪、万能试验机、差示扫描量热分析、动态力学、热重分析、旋转流变仪等测试表征方法对共混材料的增塑效果、力学性能、热行为、流变行为进行分析。实验结果表明,PEG可有效增塑PLA,PEG相对分子质量越低增塑效果越好,可以使PLA的塑化时间从250 s降低到128 s;加入PEG后,共混物的拉伸强度下降,断裂伸长率提高,PEG相对分子质量越低,拉伸强度下降越明显;PEG的加入使PLA的T_g和T_(cc)降低20℃左右,而T_m有所提高,其中低相对分子质量PEG可以更好地促进PLA结晶,但是随着PEG的加入共混体系的热分解温度降低,相对分子质量越低,热分解温度降低越明显;流变实验表明共混体系的复数黏度(η*)、储能模量(G')及损耗模量(G')的变化随PEG相对分子质量的减小下降越明显。     

Tags for the stable isotopic labeling of carbohydrates and quantitative analysis by mass spectrometry

Although stable isotopic labeling has found widespread use in the proteomics field, its application to carbohydrate quantification has been limited. Herein we report the design, synthesis, and application of a novel series of compounds that allow for the incorporation of isotopic variation within glycan structures. The novel feature of the compounds is the ability to incorporate the isotopes in a controlled manner, allowing for the generation of four tags that vary only in their isotopic content. This allows for the direct comparisons of three samples or triplicate measurements with an internal standard within one mass spectral analysis. Quantitation of partially depolymerized glycosaminoglycan mixtures, as well as N-linked glycans released from fetuin, is used to demonstrate the utility of the tetraplex tagging strategy.     

Identification and quantitative studies of protein immobilization sites by stable isotope labeling and mass spectrometry

A method was developed for characterizing immobilization sites on a protein based on stable isotope labeling and MALDI-TOF mass spectrometry. The model for this work was human serum albumin (HSA) immobilized onto silica by the Schiff base method. The immobilized HSA was digested by various proteolytic enzymes in the presence of normal water, while soluble HSA was digested in (18)O-enriched water for use as an internal standard. These two digests were mixed and analyzed, with the (18)O/(16)O ratio for each detected peptide then being measured. Several peptides in the tryptic, Lys-C, and Glu-C digests gave significantly higher (18)O/(16)O ratios than other peptides in the same digests, implying their involvement in immobilization. Analysis of these results led to identification of the N-terminus and several lysines as likely immobilization sites for HSA (e.g., K4, K41, K190, K225, K313, and K317). It was also possible from these results to quantitatively rank these sites in terms of the relative degree to which each might take part in immobilization. This method is not limited to HSA and silica but can be used with other proteins and supports.     

Poly(ethylene glycol) hydrogel-encapsulated fluorophore-enzyme conjugates for direct detection of organophosphorus neurotoxins

A simple approach is described for preparing poly-(ethylene glycol) hydrogel materials with encapsulated seminapthofluorescein (SNAFL)-organophosphorus hydrolase enzyme conjugates. Direct determination of enzyme-catalyzed neurotoxin hydrolysis is provided by the self-referencing, pH-sensitive dye SNAFL-1, whose emission spectrum changes at lambda = 550 in response to pH. Using spectrofluorimetry and paraoxon as a model organophosphate, paraoxon concentrations as low as 8 x 10(-7) M could be readily detected. On the basis of the signal-to-noise ratio, a detection limit of 16 nM was determined. The materials demonstrated high stability against enzyme-denaturing, leaching, and photobleaching when stored under ambient conditions.     

Metabolic labeling of mammalian organisms with stable isotopes for quantitative proteomic analysis

To quantify proteins on a global level from mammalian tissue, a method was developed to metabolically introduce 15N stable isotopes into the proteins of Rattus norvegicus for use as internal standards. The long-term metabolic labeling of rats with a diet enriched in 15N did not result in adverse health consequences. The average 15N amino acid enrichments reflected the relative turnover rates in the different tissues and ranged from 74.3 mpe in brain to 92.2 mpe in plasma. Using the 15N-enriched liver as a quantitative internal standard, changes in individual protein levels in response to cycloheximide treatment were measured for 310 proteins. These measurements revealed 127 proteins with altered protein level (p < 0.05). Most proteins with altered level have previously reported functions involving xenobiotic metabolism and protein-folding machinery of the endoplasmic reticulum. This approach is a powerful tool for the global quantitation of proteins, is capable of measuring proteome-wide changes in response to a drug, and will be useful for studying animal models of disease.     

聚乳酸-聚乙二醇-聚乳酸/聚(N-乙烯基吡咯烷酮)交联共聚物膜的制备与性能

在制备聚乳酸-聚乙二醇-聚乳酸(PLA-PEG-PLA)嵌段共聚物的基础上,以丙烯酸酯封端制备了PLA-PEG-PLA大分子单体。PLA-PEG-PLA大分子单体作为交联剂与N-乙烯基吡咯烷酮(NVP)单体经光聚合制备了系列PLA-PEGPLA/PVP交联共聚物膜。核磁共振测试结果表明了大分子单体的成功合成;随着NVP投料量的增加,交联共聚物膜的亲水性增强,水解降解性加快,而储能模量(E’)及玻璃化转变温度(Tg)下降;NVP与PLA-PEG-PLA大分子单体的投料量由0.25/1.68(质量比)增至1.55/1.68时,共聚膜的拉伸强度由32.7 MPa降为17.1 MPa,而断裂伸长率由82%增至387%,说明共聚物膜的韧性较好。     

Effect of poly (ethylene glycol) molecular weight and microparticle size on oral insulin delivery from P(MAA-g-EG) microparticles

Five years of successful work in our lab have shown that graft copolymer networks of poly(methacrylic acid-g-ethylene) [P(MAA-g-EG)], are very promising candidates for oral drug delivery. In an acidic environment, these copolymers form interpolymer complexes, protecting the active agent from the harsh environment of the gastrointestinal tract. At high pH, these complexes dissociate, causing the polymer to swell and release the drug. Films of P(MAA-g-EG) with a monomer ratio of 1:1 (MAA:EG) were prepared by free radical solution UV-polymerization, washed in order to remove the unreacted monomer, and crushed to form microparticles with different particle size distribution. Previous studies in our lab have focused on using polymer disks in their swelling studies. The swelling properties of polymer disks vs. crushed particles were investigated via equilibrium swelling experiments in this study. Another goal in this study is to compare different PEG chain length (MW-400 and MW-1000) and different particle size (150-212 microns, 90-150 microns and 25-90 microns) in their loading and release behavior. After 6 hours of exposing the polymer with the insulin solution we achieved approximately 90% of insulin loading.     

四臂星形聚乙二醇-苯甲酸雌二醇大分子前药的合成与表征

采用哌啶氧化物自由基氧化法将四臂星形聚乙二醇(s PEG,Mn=4400)的端羟基氧化为端羧基,合成得到了一种端羧基四臂星形聚乙二醇(s PEG-CA),并将其对苯甲酸雌二醇(EB)的17位羟基进行酯化修饰,得到四臂星形聚乙二醇-苯甲酸雌二醇大分子前药(s PEG-EB),用红外光谱和核磁共振确认了各步合成产物的结构。研究结果表明,与EB原药相比,s PEG-EB在水中的有效溶解度提高了约1630倍。s PEG-EB在p H 7.2的模拟体液中的释药试验表明,其在0~2 d内具有良好的零级释药特征,2 d时的累积释药率达到61%,5 d时的累积释药率则可达96.5%,说明s PEG-CA适合作为注射用EB的大分子前药载体。     

Correlation of relative abundance ratios derived from peptide ion chromatograms and spectrum counting for quantitative proteomic analysis using stable isotope labeling

In this study, S. cerevisiae crude membrane fractions were prepared using the acid-labile detergent RapiGest from cells grown under rich and minimal media conditions using 14N and 15N ammonium sulfate as the sole nitrogen source. Four independent MudPIT analyses of 1:1 mixtures of sample were prepared and analyzed via quantitative multidimensional protein identification technology on a two-dimensional ion trap mass spectrometer. Using the method described in this study, low-abundance integral membrane proteins with up to 14 transmembrane domains were identified and their protein expression determined when sufficient spectrum counting and ion chromatogram information was generated. We demonstrate that spectrum counting and mass spectrometry derived ion chromatograms strongly correlate for determining quantitative changes in protein expression. Spectrum counting proved more reproducible and has a wider dynamic range contributing to the deviation of the two quantitative approaches from a perfect positive correlation.     

以聚苯乙烯磺酸钠接枝聚乙二醇为模板的聚3,4-二氧乙烯噻吩水分散体的制备及性能

以苯乙烯磺酸钠(SSNa)和烯丙基聚乙二醇(APEG)为原料,制备了不同结构的聚苯乙烯磺酸钠接枝聚乙二醇(P(SS-APEG))共聚物,并以此为模板制备了聚3,4-二氧乙烯噻吩∶聚(苯乙烯磺酸钠-烯丙基聚乙二醇)(PEDOT∶P(SS-APEG))水分散体;研究了聚乙二醇(PEG)链段长度对PEDOT∶P(SS-APEG)结构与性能的影响。结果表明,通过自由基共聚,成功制备了聚苯乙烯磺酸钠接枝聚乙二醇(P(SS-APEG))共聚物。以P(SS-APEG)为模板时,EDOT的聚合速率加快,分散体粒径随APEG相对分子质量的增加而增大,水分散体表面张力减小。PEDOT薄膜的方块电阻明显降低,且APEG的相对分子质量越小,薄膜的方块电阻越低,导电性越好。当APEG的相对分子质量为700,n(SSNa)∶n(APEG)=32∶1,m(EDOT)∶m(P(SS-APEG))=1∶3时,PEDOT∶P(SS-APEG)薄膜的方块电阻较PEDOT∶PSS(m(EDOT)∶m(PSS)=1∶3)薄膜下降了3倍。