High throughput tryptic digestion via poly (acrylamide-co-methylenebisacrylamide) monolith based immobilized enzyme reactor

A poly (acrylamide-co-methylenebisacrylamide) (poly (AAm-co-MBA)) monolith was prepared by thermal polymerization in the 100 or 250 μm i.d. capillary. The monolithic support was activated by ethylenediamine followed by glutaraldehyde. Trypsin was then introduced to form an immobilized enzyme reactor (IMER). The prepared IMER showed a reliable mechanical stability and permeability (permeability constant K = 2.65 × 10⁻¹³ m²). With BSA as the model protein, efficient digestion was completed within 20 s, yielding the sequence coverage of 57%, better than that obtained from the traditional in-solution digestion (42%), which took about 12 h. Moreover, BSA down to femtomole was efficiently digested by the IMER and positively identified by matrix assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). To test the applicability of IMER for complex sample profiling, proteins extracted from Escherichia coli were digested by the IMER and further analyzed by nanoreversed phase liquid chromatography-electrospray ionization-mass spectrometry (nanoRPLC-ESI-MS/MS). In comparison to in-solution digestion, despite slightly fewer proteins were positively identified at a false discovery rate (FDR) of ∼1% (333 vs 411), the digestion time used was largely shortened (20 s vs 24 h), implying superior digestion performance for the high throughput analysis of complex samples.     

Digests or extracts?—Some interesting but conflicting results for three widely differing polluted sediment samples

Three sets of samples have been investigated in some detail. One set is from a river polluted by mine workings, containing substantial levels of Fe, Mn,Cu, Zn and Ni with traces of many other metals. The second set consists of typical estuarine sediments contaminated from a wide range of industrial sources, and the third set consists of oily drilling cuttings from the sea bottom in the vicinity of a North Sea oil production platform.These samples have been subjected to treatment 1) with EDTA at two different pH's (extracts) 2) with HNO₃/H₂O₂ 3) with HNO₃/HCl and 4) with HNO₃/HCl/HF (digests). EDTA recoveries, compared toaqua regia digests, are often very reproducible, not dependent on pH, and usually significantly low. Nitric/peroxide andaqua regia digests often give very close results suggesting that these are meaningful values indicating the maximum levels of polluting metals in the sediments. However, the triple acid digest with HF does sometimes give higher values (and reasonable agreement for CRM's such as MESS-1) but with poorer reproducibility.     

Effect of tetrahydrofuran on the conductance and ion-pairing of hydrogen chloride in wet and dry methanol mixtures

The molar conductance of dilute solutions of HCl in wet (68.5% methanol + 31.5% tetrahydrofurane mixtures at 25°C have been measured. The data were analyzed using the Fuoss-Hsia equation to calculate the infinite dilution molar conductances and association constants. The trend of the limiting conductances in these mixtures as a function of the water content shows, once more, the peculiar minimum due to the anomalous proton conductance mechanism. From these data the limiting molar conductance in the anhydrous binary solvent system was evaluated. The percentage excess proton mobility with respect to potassium ion has also been determined. All these data are compared to those found in a binary isodielectric methanol mixture containing as cosolvent 1,4-dioxane. This comparison shows that proton mobilities are very similar in both solvent mixtures. The dielectric constants, refractive indices, viscosities and densities of the methanol-tetrahydrofuran mixtures in the whole mole fraction range have been measured and are reported. An analysis of the excess molar volumes and viscosities shows a slight deviation of this system from ideality.     

A new procedure for bovine milk digestion in a focused microwave oven: gradual sample addition to pre-heated acid

Milk samples can be efficiently digested using a focused microwave oven, however the conventional procedure of addition of concentrated acids to the liquid sample leads to digestates with elevated acidity and residual carbon concentrations. In this work a focused microwave oven was applied for acid digestion of bovine milk samples using a conventional and an alternative procedure based on gradual sample addition to hot and concentrated acids. A two-level 2³ full factorial design experiment with eight runs was carried out to evaluate the optimum experimental conditions for reducing both the residual carbon and the final acidity of digestates. The three studied parameters were: temperature of the digestion medium for sample addition, addition of sulfuric acid before the sample or during the first step, and number of aliquots of the sample gradually added. The best conditions were attained by adding small aliquots of milk (ten-fold a volume of 0.5 ml added during 5.0 min) to a digestion mixture containing 3.0 ml nitric acid plus 1.0 ml sulfuric acid heated at 105 °C. It was demonstrated that the digestion efficiency of the alternative procedure was better than the conventional procedure, i.e. 98 and 80%, respectively. The alternative procedure was applied for determination of Ba, Ca, Cu, K, Mg, Na, P, and Zn in whole and non-fat bovine milk. The accuracy was proved using two certified reference materials (whole and non-fat milk powder).     

Effect of HF addition on the microwave-assisted acid-digestion for the determination of metals in coal by inductively coupled plasma-atomic emission spectrometry

The microwave-assisted acid-digestion for the determination of metals in coal by ICP-AES was investigated, especially focusing on the necessity of adding HF. By testing five certified reference materials, BCR-180, BCR-040, NIST-1632b, NIST-1632c, and SARM-20, it was found that the two-stage digestion without HF (HNO₃ + H₂O₂ was used) was very effective for the pretreatment of ICP-AES measurement. Both major metals (Al, Ca, Fe, and Mg) and minor or trace metals (Co, Cr, Cu, Mn, Ni, Pb, and Zn) in coal gave good recoveries for their certified or reference values. The possibility of ‘HF-memory effect’ was cancelled by the use of a set of vessels which had been never contacted with HF. Twenty-four Japanese standard coals (SS coals) were analyzed by the present method, and the concentrations of major metals measured by the present method provided very high accordance with those from the authentic JIS (Japanese Industrial Standard) method.     

Dynamic Effect of Operational Regulation on the Mesophilic BioMethanation of Grape Marc

Wine production annually generates an estimated 11 million metric tonnes of grape marc (GM) worldwide. The diversion of this organic waste away from landfill and towards its use in the generation of renewable energy has been investigated. This study aimed to evaluate the effectiveness of operational parameters relating to the treatment regime and inoculum source in the extraction of methane from GM under unmixed anaerobic conditions at 35 °C. The study entailed the recirculation of a previously acclimated sludge (120 days) as downstream inoculum, an increased loading volume (1.3 kg) and a low substrate-to-inoculum ratio (10:3 SIR). The results showed that an incorporation of accessible operational controls can effectively enhance cumulative methane yield (0.145 m³ CH₄ kg⁻¹ VS), corresponding to higher amounts of digestible organics converted. The calculated average volumetric methane productivity equalled 0.8802 L CH₄ L_Work⁻¹ d⁻¹ over 33.6 days whilst moderate pollutant removal (43.50% COD removal efficiency) was achieved. Molecular analyses identified Firmicutes and Bacteroidetes phyla as core organisms for hydrolytic and fermentative stages in trophic relationships with terminal electron acceptors from the methane-producing Methanosarcina genus. Economic projections established that the cost-effective operational enhancements were sustainable for valorisation from grape marc by existing wineries and distilleries.     

TiO_2-CeO_2催化剂湿式氧化苯酚的活性研究

采用共沉淀法制备了湿式氧化降解苯酚的TiO2-CeO2催化剂,测定了反应过程中不同反应时间的COD去除率,结果表明,当Ti、Ce摩尔比为3∶1时,在150℃、总压5MPa(其中氧分压4.5MPa)时,所制备的催化剂活性最高,反应90分钟COD去除率可以达到71%.对催化剂进行了比表面积、XRD和XPS测试,结果表明,高效的催化剂具有较多的活性位、催化剂粉末体系Ce的变价特性以及大量的表面吸附氧.     

A Preliminary Study on Preparation of the Aromatic/Aliphatic Co‐polyurea as Spun Fibers

Aromatic‐aliphatic co‐polyurea has been synthesized from 4,4 prime‐diphenylmethane diisocyanate (MDI), m‐phenylene diamine (m‐PDA), and 1,6‐diaminohexane (HDA) in DMAc by solution polymerization. The chemical structure of the co‐polyurea has been characterized by ¹H‐NMR. The thermal properties of the copolymers were measured by DSC and TGA. The co‐polyurea solutions were spun into fibers by means of wet spinning. The effects of coagulation conditions on the morphologies and mechanical properties of the co‐polyurea as spun fibers are discussed.     

Dephosphorylation of intact glycoprotein to greatly improve digestion efficiency coupled with matrix-assisted laser desorption/ionization–Fourier transform ion cyclotron resonance mass spectrometric analysis

Sialylation is essential for a variety of cellular functions. Herein, we used bovine fetuin with three potential N-linked glycosylation sites containing complex-type glycan structures, four potential O-linked glycosylation sites and six potential phosphorylation sites as a model compound to develop a highly-efficient digestion strategy for sialylated glycoproteins and efficient enrichment strategy for sialylated glycopeptides using titanium dioxide. The former according to the process of alkaline phosphatase digestion followed by tryptic digestion and then proteinase K digestion could greatly improve the enzymatic efficiency on fetuin, and the latter could obviously enhance the enrichment efficiency for multisialylated glycopeptides using phosphoric acid solution as elution buffer. The mass spectra of the enriched glycopeptides derived from fetuin reveal that several series of the ion clusters with mass difference of 291 Da correspond to the presence of multisialylated glycopeptides. In addition, the approach was applied to characterize the sialylated status of α2-macroglobulin and transferrin, respectively, from the sera of healthy subjects and sex- and age-matched patients with thyroid cancer, and their spectra indicate that the change in the amount of the glycoforms containing different number of sialic acid (SA) residues from one glycosylation site may be used to differentiate between healthy subjects and cancer cases.     

Differential isotope dansylation labeling combined with liquid chromatography mass spectrometry for quantification of intact and N-terminal truncated proteins

The N-terminal amino acids of proteins are important structure units for maintaining the biological function, localization, and interaction networks of proteins. Under different biological conditions, one or several N-terminal amino acids could be cleaved from an intact protein due to processes, such as proteolysis, resulting in the change of protein properties. Thus, the ability to quantify the N-terminal truncated forms of proteins is of great importance, particularly in the area of development and production of protein-based drugs where the relative quantity of the intact protein and its truncated form needs to be monitored. In this work, we describe a rapid method for absolute quantification of protein mixtures containing intact and N-terminal truncated proteins. This method is based on dansylation labeling of the N-terminal amino acids of proteins, followed by microwave-assisted acid hydrolysis of the proteins into amino acids. It is shown that dansyl labeled amino acids are stable in acidic conditions and can be quantified by liquid chromatography mass spectrometry (LC–MS) with the use of isotope analog standards.