Direct Analysis of Proteins from Solutions with High Salt Concentration Using Laser Electrospray Mass Spectrometry

The detection of lysozyme, or a mixture of lysozyme, cytochrome c, and myoglobin, from solutions with varying salt concentrations (0.1 to 250 mM NaCl) is compared using laser electrospray mass spectrometry (LEMS) and electrospray ionization-mass spectrometry (ESI-MS). Protonated protein peaks were observed up to a concentration of 250 mM NaCl in the case of LEMS. In the case of ESI-MS, a protein solution with salt concentration >?0.5 mM resulted in predominantly salt-adducted features, with suppression of the protonated protein ions. The constituents in the mixture of proteins were assignable up to 250 mM NaCl for LEMS and were not assignable above a NaCl concentration of 0.5 mM for ESI. The average sodium adducts (< n >) bound to the 7+ charge state of lysozyme for LEMS measurements from salt concentrations of 2.5, 25, 50, and 100 mM NaCl are 1.71, 5.23, 5.26, and 5.11, respectively. The conventional electrospray measurements for lysozyme solution containing salt concentrations of 0.1, 1, 2, and 5 mM NaCl resulted in < n > of 2.65, 6.44, 7.57, and 8.48, respectively. LEMS displays an approximately two orders of magnitude higher salt tolerance in comparison with conventional ESI-MS. The non-equilibrium partitioning of proteins on the surface of the charged droplets is proposed as the mechanism for the high salt tolerance phenomena observed in the LEMS measurements.

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Ambient Molecular Analysis of Biological Tissue Using Low-Energy,Femtosecond Laser Vaporization and Nanospray Postionization Mass Spectrometry

Direct analysis of plant and animal tissue samples by laser electrospray mass spectrometry (LEMS) was investigated using low-energy, femtosecond duration laser vaporization at wavelengths of 800 and 1042 nm followed by nanospray postionization. Low-energy (<50 μJ), fiber-based 1042 nm LEMS (F-LEMS) allowed interrogation of the molecular species in fresh flower petal and leaf samples using 435 fs, 10 Hz bursts of 20 pulses from a Ytterbium-doped fiber laser and revealed comparable results to high energy (75–1120 μJ), 45 fs, 800 nm Ti:Sapphire-based LEMS (Ti:Sapphire-LEMS) measurements. Anthocyanins, sugars, and other metabolites were successfully detected and revealed the anticipated metabolite profile for the petal and leaf samples. Phospholipids, especially phosphatidylcholine, were identified from a fresh mouse brain section sample using Ti:Sapphire-LEMS without the application of matrix. These lipid features were suppressed in both the fiber-based and Ti:Sapphire-based LEMS measurements when the brain sample was prepared using the optimal cutting temperature compounds that are commonly used in animal tissue cryosections.

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具有疏水核/亲水壳的双亲胶体粒子的制备

制备了具有疏水性聚苯乙烯核/亲水性聚丙烯酰胺壳的双亲粒子.疏水核通过超浓乳液聚合制备,亲水壳层通过过氧化羟基异丙苯和硫酸亚铁的界面引发制备.控制条件可得到网孔(半包覆)、褶皱(全包覆)两种形态的壳层.壳层孔的存在使得核层聚合物能够与外界接触.粒子的双亲性通过吸水吸油率进行表征.     

飞秒脉冲作用下氯丙烯的多光子解离和电离动力学

The dissociation and photoionization dynamics of C₃H₅Cl were studied at 200, 400, and 800 nm with femtosecond laser pulses. The time-of-flight mass spectra, laser power index and photoelectron images were recorded. At short wavelength (200 nm), ionization of the parent molecule was found to be the dominant channel, while other ions were generated by the dissociation of C₃H₅Cl⁺. With the shift to long wavelength (e.g., 800 nm), fragment ions became dominant, and were generated through the multiphoton ionization of neutral fragments after the photodissociation of C₃H₅Cl. These results imply that photodissociation plays a significant role at long wavelength, because neutral fragments are supposed to be generated from the intermediate states reached by 800 nm photons. At 400 nm, the dissociation on the intermediate states is also critical, but is not as high as that at 800 nm. Taken together, our results demonstrate that the dissociation/ionization behaviors of allyl chloride are wavelength-dependent, and reveal the complex dynamics of allyl chloride at 200, 400 and 800 nm.     

Nano-Encapsulation of Proteins Via Self-Assembly with Lipids and Polymers

Summary: The solubilization and encapsulation of the weakly soluble protein hemoglobin was investigated at the nanoscale using self-assembly with the branched polymer polyethyleneimine (PEI), the lipid glycerol monooleate (GMO), and two amphiphilic poly(ethylenglycol) monooleate derivatives with molecular weights 2100 g/mol (MO-PEG1) and 860 g/mol (MO-PEG2). The created self-assembly nanovehicles were analyzed by quasi-elastic light scattering (QELS) in order to determine their sizes as well as by circular dichroism in order to characterize the protein presence in the nanoobjects. The cationic polymer PEI formed mixed nano-objects with the protein hemoglobin. The polymer conformation in the nanovehicle was established to be sensitive to dilution, a property that can be essential for the protein release upon administration. The amphiphile MO-PEG1 was a co-surfactant in the dispersion of monoglyceride lipid nanoobjects needed for the hemoglobin encapsulation. The amphiphile MO-PEG2 formed small micelles in the absence of a lipid. The nanoobjects dispersions were studied for their stability on storage and reproducibility.     

Preparative Hydrophobic Interaction Chromatography of Proteins Using Ether Based Chemically Bonded Phases

Abstract

This paper examines the use of 15–20 micron wide-pore silica-based ether bonded phases for the preparative hydrophobic interaction chromatography of proteins. In particular, silyl ethers are immobilized on large particle silica in an analogous manner to previously developed ether bonded 5 um analytical supports. The preparative supports are reproducibly prepared and exhibit constant chromatographic retention for at least five months of continual use. Preparative columns can be operated for protein chromatography with peak shapes and capacity as predicted by the Snyder gradient elution model. Moreover, similar retention times are obtained relative to those on the 5 um analytical columns, enabling the direct transition and scale-up of separation. Gradient optimization is seen to directly parallel that performed on 5 um bonded ether analytical columns. Acceptable chromatographic resolution was obtained with sample capacity of >15 mg protein/ml column volume using a repetitive injection technique. A column clean-up strategy is examined for rapid and safe removal of contaminants. An illustrative example of use of the bonded ether preparative columns is made by application to soybean trypsin inhibitor purification. Initial results are presented on a column-switching method for the analytical monitoring of preparative separation.     

Hydrophobic Interaction Chromatography of t-RNA's and Proteins

Abstract

Various microparticulate siliceous bonded stationary phases having weakly hydrophobic ligates were developed for HPLC of proteins and t-RNA's by hydrophobic interaction chromatography (HIC). It was confirmed that optimal separation of different types of biopolymers can be obtained by using a set of stationary phases having appropriate hydrophobic properties. Thus, the separation of t-RNA's is best carried out on stationary phases which are more hydrophobic than those optimal for HIC of proteins. Plots of log k' of both proteins and t-RNA's against the salt molality in the eluent yielded straight lines at sufficiently high salt concentrations in the eluent. The limiting slopes represent the hydrophobic interaction parameter for the particular chromatographic system and can serve as measures of the hydrophobic character of either the biopolymer or the stationary phase. Stationary phases with covalently bound polyether chains at the surface were found to be most suitable for HIC of proteins and t-RNA's.     

Direct Analysis of Triterpenes from High-Salt Fermented Cucumbers Using Infrared Matrix-Assisted Laser Desorption Electrospray Ionization (IR-MALDESI)

High-salt samples present a challenge to mass spectrometry (MS) analysis, particularly when electrospray ionization (ESI) is used, requiring extensive sample preparation steps such as desalting, extraction, and purification. In this study, infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) coupled to a Q Exactive Plus mass spectrometer was used to directly analyze 50-μm thick slices of cucumber fermented and stored in 1 M sodium chloride brine. From the several hundred unique substances observed, three triterpenoid lipids produced by cucumbers, β-sitosterol, stigmasterol, and lupeol, were putatively identified based on exact mass and selected for structural analysis. The spatial distribution of the lipids were imaged, and the putative assignments were confirmed by tandem mass spectrometry performed directly on the same cucumber, demonstrating the capacity of the technique to deliver confident identifications from highly complex samples in molar concentrations of salt without the need for sample preparation.

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Simultaneous Recovery of Lipids and Proteins by Enzymatic Hydrolysis of Fish Industry Waste Using Different Commercial Proteases

Four different commercial proteases (Protease-P-Amano6, Alcalase^®, Protex 7L^®, and Neutrase^®) were evaluated for recovering lipids and protein simultaneously by hydrolysis. Fungal protease (Protease-P-Amano6) resulted in maximum lipid recovery (74.9%) followed by alcalase (61.7%). Peroxide value (PV; milli-equivalents of oxygen per kilogram) in the oil recovered after hydrolysis was 40.48 compared to 8.7 in lipids from fresh fish viscera. However, addition of tertiary butyl hydroxyl quinine at 200 ppm level maintained the PV of oil recovered by hydrolysis closer to oil from fresh waste. Degree of hydrolysis was the highest in case of fungal protease (49.1%) where neutrase resulted in higher total antioxidant activity (micrograms of ascorbic acid equivalents per milligram protein) of 34.4. Protein hydrolysate prepared using fungal protease had the higher diphenylpicrylhydrazyl radical scavenging activity as compared to those from other enzymes. The results indicate the utility of commercial proteases in providing an ecofriendly and feasible solution for reducing disposal problems associated with fish processing.     

Analysis of Fragmentation Pathways of New-Type Synthetic Cannabinoids Using Electrospray Ionization

Recently, dozens of new psychoactive substances have appeared on the European drug market every year. The most abundant group of these compounds is synthetic cannabinoids. In the first few years of the “legal highs” phenomenon, JWH (John W. Huffman) compounds were especially popular among drug users. However, the group of synthetic cannabinoids is constantly expanding, as new compounds are created by replacing known structural elements with different chemical groups. The problem with the identification of novel substances in forensic laboratories results from the structural similarity of the compounds and the rapid introduction of newer designer drugs on the black market. In this study, the fragmentation patterns of 29 new-type synthetic cannabinoids using electrospray ionization were investigated. The analysis was performed using quadrupole time-of-flight mass spectrometry. Based on measurements carried out under various conditions, the way of fragmentation of the tested compounds that were divided into groups due to their chemical structure was established. The study showed that the bond between the carbon atom of the carbonyl group and the ring or NH group attached to the ring was mainly cleaved. This mechanism was adequate for the fragmentation of first-generation synthetic cannabinoids. This paper presents characteristic ions formed by synthetic cannabinoids (i.e., ions originating from an indole/indazole ring and an adamanyl/naphthalene/quinoline ring) using electrospray ionization. Knowledge of these specific fragments can be used in forensic laboratories to determine the structure of novel compounds from the group of synthetic cannabinoids.

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UV Lamp as a Facile Ozone Source for Structural Analysis of Unsaturated Lipids Via Electrospray Ionization-Mass Spectrometry

Ozonolysis of alkene functional groups is a type of highly specific and effective chemical reaction, which has found increasing applications in structural analysis of unsaturated lipids via coupling with mass spectrometry (MS). In this work, we utilized a low-pressure mercury lamp (6 W) to initiate ozonolysis inside electrospray ionization (ESI) sources. By placing the lamp near a nanoESI emitter that partially transmits 185 nm ultraviolet (UV) emission from the lamp, dissolved dioxygen in the spray solution was converted into ozone, which subsequently cleaved the double bonds within fatty acyls of lipids. Solvent conditions, such as presence of water and acid solution pH, were found to be critical in optimizing ozonolysis yields. Fast (on seconds time scale) and efficient (50%–100% yield) ozonolysis was achieved for model unsaturated phospholipids and fatty acids with UV lamp-induced ozonolysis incorporated on a static and an infusion nanoESI source. The method was able to differentiate double bond location isomers and identify the geometry of the double bond based on yield. The analytical utility of UV lamp-induced ozonolysis was further demonstrated by implementation on a liquid chromatography (LC)-MS platform. Ozonolysis was effected in a flow microreactor that was made from ozone permeable tubing, so that ambient ozone produced by the lamp irradiation could diffuse into the reactor and induce online ozonolysis post-LC separation and before ESI-MS.

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牛油果中化学成分的内部萃取电喷雾电离质谱检测

采用内部萃取电喷雾电离质谱(iEESI-MS)技术,以甲醇作为萃取溶剂,无需样品预处理,在优化条件下分别对牛油果果肉和果皮中化学成分进行快速直接鉴定,并考察了不同成熟度牛油果果肉中化学成分的差异.实验结果表明,在负离子检测模式下,从果肉中鉴定出棕榈酸、硬脂酸、棕榈油酸、油酸和亚麻酸等12种有效营养成分,从果皮中鉴定出儿茶素、绿原酸等11种营养成分.利用正交偏最小二乘判别分析法(OPLS-DA)对不同成熟度的牛油果果肉的iEESI-MS指纹谱图数据进行分析发现,此方法能够有效判别不同成熟程度的牛油果且5种化学成分差异显著.本方法无需样品预处理、样品耗量少、分析速度快(单个样品检测时间小于1 min)且操作简便,为植物有效营养成分和医用价值的开发提供了一种快速质谱分析新方法.     

Isothermal Titration Microcalorimetric Studies of the Effect of Temperature on Hydrophobic Interaction between Proteins and Hydrophobic Adsorbents

This study attempted to comprehend how temperature affects hydrophobic interaction between proteins and hydrophobic adsorbents. By equilibrium batch analysis, we measured the adsorption isotherm to evaluate the protein-adsorbent affinity, while isothermal titration calorimetry was used to measure the adsorption enthalpy. In addition, the affinity and enthalpy differences between two proteins, alpha-chymotrypsinogen A and trypsinogen, with two adsorbents, butyl-Sepharose and octyl-Sepharose gel, under varying temperatures were studied with respect to the exposed hydrophobic segments of the protein and ligand hydrophobicity. The enthalpies obtained in this investigation can be used to more thoroughly understand the hydrophobic interaction between proteins and adsorbents. First, the adsorption isotherm experiments reveal that the adsorption quantity of the proteins with the Sepharose gels increases with temperature. For a microcalorimetric measurement, as temperature is increased from 298 to 310 K, the DeltaH value of alpha-chymotrypsinogen A with butyl-Sepharose increases, while the DeltaH value of trypsinogen is reduced. This is likely due to the fact that alpha-chymotrypsinogen A has a higher area of exposed hydrophobic segments than trypsinogen does. This observation also implies that as temperature increases, the interaction mechanism of alpha-chymotrypsinogen A with butyl-Sepharose changes from an adsorption-dominated process to a partitioning process. In addition, for octyl-Sepharose, the DeltaH value of alpha-chymotrypsinogen A is positive and decreases with temperature increment. However, the DeltaH value of trypsinogen was positive and increased with temperature. Therefore, we conclude that as temperature increases, the interaction mechanism of the proteins for octyl-Sepharose is a partitioning-dominated process. Copyright 2000 Academic Press.     

Profiling Metal Oxides with Lipids: Magnetic Liposomal Nanoparticles Displaying DNA and Proteins

Metal oxides include many important materials with various surface properties. For biomedical and analytical applications, it is desirable to engineer their biocompatible interfaces. Herein, a phosphocholine liposome (DOPC) and its headgroup dipole flipped counterpart (DOCP) were mixed with ten common oxides. Using the calcein leakage assay, cryo‐TEM, and ζ‐potential measurement, these oxides were grouped into three types. The type 1 oxides (Fe₃O₄, TiO₂, ZrO₂, Y₂O₃, ITO, In₂O₃, and Mn₂O₃) form supported bilayers only with DOCP. Type 2 (SiO₂) forms supported bilayers only with DOPC; type 3 (ZnO and NiO) are cationic and damage lipid membranes. Magnetic Fe₃O₄ nanoparticles were further studied for conjugation of fluorophores, proteins, and DNA to the supported DOCP bilayers via lipid headgroup labeling, covalent linking, or lipid insertion. Delivery of the conjugates to cells and selective DNA hybridization were demonstrated. This work provides a general solution for coating the type 1 oxides with a simple mixing in water, facilitating applications in biosensing, separation, and nanomedicine.     

基质辅助激光解析电离-串联飞行时间质谱与电喷雾离子化-四极杆-飞行时间质谱分析蛋白质

为比较基质辅助激光解析电离-串联飞行时间质谱(MALDI-TOF-TOF)和电喷雾离子化-四极杆-飞行时间质谱(ESI-Q-TOF)在蛋白质定性和定量分析方面的性能,分别利用两种仪器对用于相对和绝对定量的等量异位标签(iTRAQ)标记的雌激素刺激前后MCF7细胞内的蛋白质水解产生的多肽进行了定性和定量分析.结果表明,用MALDI-TOF-TOF和ESI-Q-TOF方法分别鉴定出1086种和848种蛋白质,其中相同的蛋白质为633种;MALDI-TOF-TOF和ESI-Q-TOF方法分别找到38种和33种表达差异在0.5倍以上的蛋白质,其中3种为相同的蛋白质.实验数据说明,两种仪器在蛋白质定性和定量分析方面有很大的互补性,将两种仪器结合使用,不仅能够显著提高蛋白质定性和定量分析的覆盖率,而且可提高分析的置信度.     

电喷雾内部萃取电离质谱直接分析蒜瓣组织的研究

固态复杂基体样品的直接电离一般仅发生在样品浅层表面,对固体内部深层物质的质谱分析往往需将样品破碎后才能进行。本研究以蒜瓣样品为例,无需样品预处理,带电的甲醇溶液以2μL/min持续流经蒜瓣深层组织,内部组织中的化学物质选择性地溶解到流动液中,并在蒜瓣组织的尖端形成电喷雾,产生相应气态离子供后续质谱分析。在正离子检测模式下,实验记录了两种蒜瓣样品(共24个)、经不同方式贮存加工后的同种蒜瓣样品(共36个)在m/z 50~2000范围内的化学指纹谱图,并将指纹谱图数据进行主成分分析( PCA),获得了令人满意的分组结果。研究表明,电喷雾内部萃取电离质谱( iEESI-MS)可以直接获取蒜瓣内部组织生物化学信息,鉴定蒜瓣组织中重要的化学成分(如蒜氨酸、蒜素、精氨酸、多糖等),快速识别蒜瓣组织中代谢组分的变化。本方法无需样品预处理、操作简单、分析速度快(单个样品分析时间小于2 min),最大程度避免了生物组织分析过程中活性物质受环境作用(如酸解、空气氧化等)的降解,有望为生物组织样品的代谢组学研究提供一种直接、快速的质谱分析方法。