Evaluation of multi-solvent size exclusion chromatography columns packed with sub-2 µm particles for the characterization of synthetic polymers.

With the recent development of small particle stationary-phases and dedicated instrumentation, the combination of size-exclusion chromatography (SEC) with ultra-high performance liquid chromatography (UHPLC) technology has been realized. It opened up a new polymer analysis technique called UHP-SEC. Although high resolution and fast analysis can be achieved, the multi-solvent suitability for a given column was limited to either organic or aqueous eluents. In this work, the capability of novel SEC columns (AdvanceBio SEC columns) packed with 1.9 µm particles for the characterization of synthetic polymers in organic solvents as well as the multi-solvent compatibility for organic and aqueous eluents have been demonstrated. About six times faster separation for both polystyrene (PS) and polyethylene glycol (PEG) with good peak shape and repeatability were achieved in comparison with standard SEC columns at comparable resolution. Especially for PEG, in contrast to other SEC columns, this column could provide close-to-accurate determination of molecular weights with tetrahydrofuran (THF) as mobile phase. Good reproducibility was obtained after switching several times from water to THF and vice versa with RSD% in retention times less than 0.5 %. Different samples such as polyols, isocyanates and additives can also be analyzed for molecular weight and distribution or composition determination. Volume overload, especially with injection volumes higher than 10 µL needs to be considered. This new column offers a powerful choice for oligomer and polymer analysis with both aqueous and organic mobile phase. Ultimately, hyphenating SEC columns to various detectors can enable more information regarding chemical composition, molecular weight, concentration, and structure.

Effect of Hydrogen on Corrosion Behavior of 321 Stainless Steel in NH4Cl Solution.

For a hydrogenation heat exchanger operating under severe working conditions such as high temperature, high pressure and a hydrogen environment, perforation accidents caused by NH4Cl corrosion occur frequently. However, few reports on the effect of hydrogen on the corrosion behavior of metal materials in NH4Cl aqueous solution have been published. In this paper, X-ray photoelectron spectroscopy (XPS), electrochemical dynamic potential polarization, electrochemical impedance spectroscopy (EIS), Mott-Schottky (M-S) curves and scanning electron microscopy (SEM) were used to study the effect of electrochemical hydrogen charging (EHC) on the corrosion behavior of 321 stainless steel in an NH4Cl solution environment. The results show that: (1) hydrogen can change the structure and chemical composition of 321 stainless steel passive film and promote the conversion of metal oxide to hydroxide. At the same time, it can reduce the stability of the passive film. (2) Hydrogen can increase the thermodynamic and kinetic tendency of corrosion reaction and cooperate with Cl- to promote the occurrence of pitting corrosion.

Influences of Ultrasonic Image-Guided Erector Spinae Plane Block on Postoperative Pulmonary Air Content of Lung Carcinoma Patients Undergoing Thoracoscopic Surgery.

To investigate the influences of ultrasonic image-guided erector spinae plane block (ESPB) on postoperative pulmonary air content of lung carcinoma patients undergoing thoracoscopic surgery, 42 patients performed with thoracoscopic radical surgery for lung carcinoma were selected. The patients in the experimental group were performed with ultrasound-guided unilateral ESPB and intravenous general anesthesia. The patients in the control group only underwent intravenous anesthesia. The changes in postoperative pulmonary air content between the two groups were compared. After that, all included patients were divided into the experimental (senior) group (13 cases), the experimental (adult) group (8 cases), the control (senior) group (11 cases), and the control (adult) group (10 cases) according to age. The changes in postoperative pulmonary air content of patients in the four groups were compared. The results showed that lung ultrasound score (LUS) of patients in experimental group was 6.4 ± 3.2 points 0.5 hour after catheter extraction and LUS was 4.1 ± 2.3 points 20 to 30 hours. Both scores were remarkably lower than those of patients in control group (P < 0.05). LUS of lower left anterior area, upper left posterior area, lower left posterior area, upper right posterior area, and lower right posterior area of patients in experimental group was all apparently lower than those in control group 0.5 hour after catheter extraction (P < 0.05). LUS of upper left posterior area, lower left posterior area, lower right anterior area, upper right posterior area, and lower right posterior area of patients in experimental group was all remarkably lower than those in control group 20 to 30 hours after surgery (P < 0.05). LUS of senile patients and middle-aged patients in experimental group 0.5 hour after catheter extraction was 8.01 ± 2.48 points and 5.93 ± 3.91 points, respectively, which were both notably lower than those in control group (P < 0.05). Ultrasound-guided ESPB exerted fewer influences on lung and could effectively improve postoperative pulmonary air content among patients. Hence, it was worthy of clinical promotion.

Conversion of calibration curves for accurate estimation of molecular weight averages and distributions of polyether polyols by conventional size exclusion chromatography.

Accurate measurement of molecular weight averages (M¯n,M¯w,M¯z) and molecular weight distributions (MWD) of polyether polyols by conventional SEC (size exclusion chromatography) is not as straightforward as it would appear. Conventional calibration with polystyrene (PS) standards can only provide PS apparent molecular weights which do not provide accurate estimates of polyol molecular weights. Using polyethylene oxide/polyethylene glycol (PEO/PEG) for molecular weight calibration could improve the accuracy, but the retention behavior of PEO/PEG is not stable in THF-based (tetrahydrofuran) SEC systems. In this work, two approaches for calibration curve conversion with narrow PS and polyol molecular weight standards were developed. Equations to convert PS-apparent molecular weight to polyol-apparent molecular weight were developed using both a rigorous mathematical analysis and graphical plot regression method. The conversion equations obtained by the two approaches were in good agreement. Factors influencing the conversion equation were investigated. It was concluded that the separation conditions such as column batch and operating temperature did not have significant impact on the conversion coefficients and a universal conversion equation could be obtained. With this conversion equation, more accurate estimates of molecular weight averages and MWDs for polyether polyols can be achieved from conventional PS-THF SEC calibration. Moreover, no additional experimentation is required to convert historical PS equivalent data to reasonably accurate molecular weight results.

Facile synthesis of C8-functionalized magnetic silica microspheres for enrichment of low-concentration peptides for direct MALDI-TOF MS analysis.

In this study, novel C8-functionalized magnetic polymer microspheres were prepared by coating single submicron-sized magnetite particle with silica and subsequent modification with chloro (dimethyl) octylsilane. The resulting C8-functionalized magnetic silica (C8-f-M-S) microspheres exhibit well-defined magnetite-core-silica-shell structure and possess high content of magnetite, which endow them with high dispersibility and strong magnetic response. With their magnetic property, the synthesized C8-f-M-S microspheres provide a convenient and efficient way for enrichment of low-abundance peptides from tryptic protein digest and human serum. The enriched peptides/proteins were subjected for MALDI-TOF MS analysis and the enrichment efficiency was documented. In a word, the facile synthesis and efficient enrichment process of the novel C8-f-M-S microspheres make them promising candidates for isolation of peptides even in complex biological samples such as serum, plasma, and urine.

Novel Fe3O4@TiO2 core-shell microspheres for selective enrichment of phosphopeptides in phosphoproteome analysis.

Due to the dynamic nature and low stoichiometry of protein phosphorylation, enrichment of phosphorylated peptides from proteolytic mixtures is often necessary prior to their characterization by mass spectrometry. Immobilized metal affinity chromatography (IMAC) is a popular way to enrich phosphopeptides; however, conventional IMAC lacks enough specificity for efficient phosphoproteome analysis. In this study, novel Fe 3O 4@TiO 2 microspheres with well-defined core-shell structure were prepared and developed for highly specific purification of phosphopeptides from complex peptide mixtures. The enrichment conditions were optimized using tryptic digests of beta-casein, and the high specificity of the Fe 3O 4@TiO 2 core-shell microspheres was demonstrated by effectively enriching phosphopeptides from the digest mixture of alpha-casein and beta-casein, as well as a five-protein mixture containing nonphosphoproteins (bovine serum albumin (BSA), myoglobin, cytochrome c) and phosphoproteins (ovalbumin and beta-casein). The Fe 3O 4@TiO 2 core-shell microspheres were further successfully applied for the nano-LC-MS/MS analysis of rat liver phosphoproteome, which resulted in identification of 56 phosphopeptides (65 phosphorylation sites) in mouse liver lysate in a single run, indicating the excellent performance of the Fe 3O 4@TiO 2 core-shell microspheres.

Novel approach for the synthesis of Fe3O4@TiO2 core-shell microspheres and their application to the highly specific capture of phosphopeptides for MALDI-TOF MS analysis.

A novel approach is proposed to synthesize Fe(3)O(4)@TiO(2) microspheres with a well-defined core-shell structure, and the synthesized Fe(3)O(4)@TiO(2) core-shell microspheres were successfully applied for the simple and fast enrichment of phosphopeptides via direct MALDI-TOF mass spectrometry analysis.

Preparation of Fe3O4@ZrO2 core-shell microspheres as affinity probes for selective enrichment and direct determination of phosphopeptides using matrix-assisted laser desorption ionization mass spectrometry.

Fe3O4@ZrO2 microspheres with well-defined core-shell structure were prepared and applied for the highly selective enrichment of phosphopeptides from tryptic digest product of proteins. To successfully coat iron oxide microspheres with uniform zirconia shell, magnetic Fe3O4 microspheres were first synthesized via a solvothermal reaction, followed by being coated with a thin layer of carbon by polymerization and carbonization of glucose through hydrothermal reaction. Finally, with the use of the Fe3O4@C microspheres as templates, zirconium isopropoxide was prehydrolyzed and absorbed onto the microspheres and eventually converted into zirconia by calcinations. The as-prepared Fe3O4@ZrO2 core-shell microspheres were used as affinity probes to selectively concentrate phosphopeptides from tryptic digest of beta-casein, casein, and five protein mixtures to exemplify their selective enrichment ability of phosphopeptides from complex protein samples. In only 0.5 min, phosphopeptides sufficient for characterization by MALDI-MS could be enriched by the Fe3O4@ZrO2 microspheres. The results demonstrate that Fe3O4@ZrO2 microspheres have the excellent selective enrichment capacity for phosphopeptides from complex samples. The performance of the Fe3O4@ZrO2 microspheres was further compared with commercial IMAC beads for the enrichment of peptides originating from tryptic digestion of beta-casein and bovine serum albumin (BSA) with a molar ratio of 1:50, and the results proved a stronger selective ability of Fe3O4@ZrO2 microspheres over IMAC beads. Finally, the Fe3O4@ZrO2 microspheres were successfully utilized for enrichment of phosphopeptides from human blood serum without any other purification procedures.

Immobilization of trypsin on superparamagnetic nanoparticles for rapid and effective proteolysis.

In this work, a novel and facile route was developed for the immobilization of enzyme on nanosized magnetic particles, and its application to fast protein digestion via a direct MALDI-TOF mass spectrometry analysis was demonstrated. At first, amine-functionalized magnetic particles with high magnetic responsivity and excellent dispersibility were prepared through a facile one-pot strategy. Then, magnetic nanoparticles were functionalized with numerous aldehyde(-CHO) groups by treating the as-synthesized, amine-functionalized magnetic nanoparticles with glutaraldehyde. Finally, immobilization of trypsin onto the aldehyde-functionalized magnetic nanoparticles was achieved through reaction of the aldehyde groups with amine groups of trypsin. The obtained trypsin-immobilized magnetic nanoparticles were conveniently applied for protein digestion. The digestion efficiency was demonstrated with peptide mapping analysis of three model proteins. The process of digestion is very facile due to the easy manipulation of magnetic nanoparticles. Complete protein digestion was achieved in a short time (5 min), without any complicated reduction and alkylation procedures. These results are expected to open up a new possibility for the proteolysis analysis as well as a new application of magnetic nanoparticles. Additionally, it is worth noting that, since the preparation and surface functionality of magnetic nanoparticles is low-cost and reproducible, the preparation method and application approach of the magnetic nanoparticles may find much potential in proteome research.

On-column tryptic mapping of proteins using metal-ion-chelated magnetic silica microspheres by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Peptide mapping analysis, utilizing an easily replaceable and regenerable on-column enzymatic microreactor with metal-ion-chelated adsorption of enzyme on magnetic silica microspheres, combined with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), was developed. Firstly, magnetic microspheres of small size and strong magnetism were prepared through solvothermal reaction. Thereafter, by introducing tetraethyl orthosilicate (TEOS), magnetic silica (MS) microspheres were formed. Trypsin could then be immobilized onto the MS microspheres based on the Lewis acid-base interaction through the divalent cation chelators such as iminodiacetic acid (IDA), which was chemically bound to the microspheres through the introduction of glycidoxypropyltrimethoxysilane (GLYMO). The trypsin-immobilized MS microspheres were then locally packed into the capillary by the application of a strong magnetic field using a magnet. The performance of the method was exemplified with digestion of bovine serum albumin for 5 min at 50 degrees C and the result was comparable to the 12 h in-solution digestion. The ability of regeneration of the prepared on-column microreactor and good reproducibility of microreactor before and after regeneration were also demonstrated.

Efficient on-chip proteolysis system based on functionalized magnetic silica microspheres.

An easily replaceable enzymatic microreactor has been fabricated based on the glass microchip with trypsin-immobilized magnetic silica microspheres (MS microspheres). Magnetic microspheres with small size (approximately 300 nm in diameter) and high magnetic responsivity to magnetic field (68.2 emu/g) were synthesized and modified with tetraethyl orthosilicate (TEOS). Aminopropyltriethoxysilane (APTES) and glutaraldehyde (GA) were then introduced to functionalize the MS microspheres for enzyme immobilization. Trypsin was stably immobilized onto the MS microspheres through the reaction of primary amines of the proteins with aldehyde groups on the MS microspheres. The trypsin-immobilized MS microspheres were then locally packed into the microchannel by the application of a strong field magnet to form an on-chip enzymatic microreactor. The digestion efficiency and reproducibility of the microreactor were demonstrated by using cytochrome c (Cyt-C) as a model protein. When compared with an incubation time of 12 h by free trypsin in the conventional digestion approach, proteins can be digested by the on-chip microreactor in several minutes. This microreactor was also successfully applied to the analysis of an RPLC fraction of the rat liver extract. This opens a route for its further application in top-down proteomic analysis.

Microchip reactor packed with metal-ion chelated magnetic silica microspheres for highly efficient proteolysis.

An easily replaceable and regenerable protease microreactor with metal-ion chelated adsorption of enzyme has been fabricated on chip. Magnetic microspheres with small size (approximately 200 nm in diameter) and strong magnetism were synthesized and were modified with tetraethyl orthosilicate. The metal chelating agent of iminodiacetic acid was then reacted with glycidoxypropyltrimethoxysilane before its immobilization onto the surface of magnetic silica microspheres (MS microspheres). The metal ion of copper and enzyme were subsequently adsorbed onto the surface. The prepared MS microspheres were then locally packed into the microchannel by the application of a strong magnetic field using a magnet to form an on-chip enzymatic microreactor. Capability of the proteolytic microreactor was demonstrated by cytochrome c and bovine serum albumin as model proteins. The digestion products were characterized using MALDI-TOF/TOF MS with sequence coverage of 77% and 21% observed, respectively. This microreactor was also applied to the analysis of one RPLC fraction of rat liver extract. After a database search, 23 unique peptides corresponding to 7 proteins were identified when one RPLC fraction of rat liver extract was digested by the microreactor. This opens a route for its future application in top-down proteomic analysis.

Comparison of 2-D LC and 3-D LC with post- and pre-tryptic-digestion SEC fractionation for proteome analysis of normal human liver tissue.

The current "shotgun" proteomic analysis, strong cation exchange-RPLC-MS/MS system, is a widely used method for proteome research. Currently, it is not suitable for complicated protein sample analysis, like mammal tissues or cells. To increase the protein identification confidence and number, an additional separation dimension for sample fractionation is necessary to be coupled prior to current multi-dimensional protein identification technology (MudPIT). In this work, SEC was elaborately selected and applied for sample prefractionation in consideration of its non-bias against sample and variety of choice of mobile phases. The analysis of the global lysate of normal human liver tissue sample provided by the China Human Liver Proteome Project, were performed to compare the proteome coverage, sequence coverage (peptide per protein identification) and protein identification efficiency in MudPIT, 3-D LC-MS/MS identification strategy with preproteolytic and postproteolytic fractionation. It was demonstrated that 3-D LC-MS/MS utilizing protein level fractionation was the most effective method. A MASCOT search using the MS/MS results acquired by QSTAR(XL) identified 1622 proteins from 3-D LC-MS/MS identification approaches. A primary analysis on molecular weight, pI and grand average hydrophobicity value distribution of the identified proteins in different approaches was made to further evaluate the 3-D LC-MS/MS analysis strategy.

Analysis of nuclear proteome in C57 mouse liver tissue by a nano-flow 2-D-LC-ESI-MS/MS approach.

The analysis of whole cell or tissue extracts is too complex for current protein identification technology and not suitable for the study of proteins with low copy levels. To concentrate and enrich low abundance proteins, organelle proteomics is a promising strategy. This approach can not only reduce the protein sample complexity but also provide information about protein location in cells, organs, or tissues under analysis. Nano-flow two-dimensional strong-cation exchange chromatography (SCX)-RPLC-ESI-MS/MS is an ideal platform for analyzing organelle extracts because of its advantages of sample non-bias, low amounts of sample required, powerful separation capability, and high detection sensitivity. In this study, we apply nano-scale multidimensional protein identification technology to the analysis of C57 mouse liver nuclear proteins. Organelle isolation has been optimized to obtain highly pure nuclei. Evaluation of nucleus integrity and purity has been performed to demonstrate the effectiveness of the optimized isolation procedure. The extracted nuclear proteins were identified by five independent nano-flow on-line SCX-RPLC-ESI-MS/MS analyses to improve the proteome coverage. Finally, a total of 462 proteins were identified. Corresponding analyses of protein molecular mass and pI distribution and biological function categorization have been undertaken to further validate our identification strategy.

Detailed studies on the enantioselective synthesis and HPLC enantioseparation of N-protected 3-hydroxyglutarimides.

An analytical HPLC method using CHIREX (S)-LEU/(S)-alpha-NEA column was developed for the determination of the enantiomeric excesses of N-protected (S)-3-hydroxyglutarimides. Using this method, detailed studies on the base-promoted ring-expansion reaction of the amidolactones, derived from l-glutamic acid, were undertaken.