Structural characterization of glycopeptides by N-terminal protein ladder sequencing

High-sensitivity and high-throughput mass spectrometry (MS) has become an important tool for characterizing glycopeptides. Here, we analyzed synthetic O-linked glycopeptides using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS. First, we applied MALDI-quadrupole ion trap (QIT)-TOF MS, which enables collision-induced dissociation-MSn analysis for fine structural characterization. Subsequent MS/MS of sodium adduct ions selected as precursor ions yielded detailed information about the site of oligosaccharide attachment as well as the carbohydrate and amino acid sequences; however, these MS/MS spectra were very complex. To obtain easily interpretable and simple spectra, we used N-terminal protein ladder sequencing coupled with MALDI-TOF MS. From the extremely simple resulting spectra, we were able to determine the glycosylation sites, amino acid sequences, and oligosaccharide molecular weights of the glycopeptides.     

Transport of human immunoglobulin G and Fc-fusion proteins to chicken egg yolk

We examined the transport of human immunoglobulin G (IgG) subclasses and fusion proteins with the Fc region of human IgG to the egg yolk, after the proteins were injected into a vein of hens. Human IgGs were efficiently transported and accumulated into the yolk, whereas the proteins were not detected in the egg white. Among human IgG subclasses, IgG2 was transported most efficiently. Fc-fusion proteins injected were also transported into the yolk. A fusion protein with the Fc region derived from human IgG2 was more efficiently transported into the yolk than the counterpart fusion with the Fc region from human IgG1. This study shows that the recovery of recombinant antibodies and Fc-fusion proteins from the yolk is an effective method in transgenic chicken bioreactors.     

Zeta potential of alumina powders with different crystalline phases in simulated body fluids

The zeta potential of alumina (Al₂O₃) powder with different crystalline phases, prepared by heat treatment of boehmite, was measured in simulated body fluids in order to discuss the mechanism on in vivo formation of a calcium and phosphorus (CaP)-rich layer on bone cement containing δ-Al₂O₃-based bead powder. γ, δ, and θ-Al₂O₃ powders were obtained by heat treatment of boehmite powder at 600 °C, 900 °C, and 1025 °C, respectively. It was found that δ-Al₂O₃ gave a negative zeta potential in an acidic simulated body fluid, whereas γ-Al₂O₃ and θ-Al₂O₃ gave a positive potentials. During the bone fracture healing process, acidic conditions are maintained at the site of fracture for several days. Consequently, it is speculated that the negative surface potential of δ-Al₂O₃ in an acidic body fluid, similar to the fracture site, might be responsible for the in vivo formation of the CaP-rich layer on the overlying bone cement, given that the negatively charged surface of δ-Al₂O₃ would attract calcium ions from the surrounding body fluid, thereby facilitating the formation of the CaP-rich layer.     

Analysis of the effects of thermal protein denaturation on the quality attributes of sous‐vide cooked tuna

Traditional (low temperature, long time) and novel (low temperature, short time) sous‐vide cooking of lean tuna were characterized by analyzing the effects of thermal protein denaturation (TPD) on quality attributes, such as color, appearance, shrinkage, drip loss, and texture. TPD was analyzed by differential scanning calorimetry and estimated for several thermal schedules by kinetic analysis, following the dynamic method. When heated at a rate of 10 °C/min, myosin began to denature at around 35 °C. Actin did not denature, even when the temperature rose to approximately 51 °C, until the denaturation of myosin was complete. However, actin began to denature at approximately 58 °C and was completely denatured at 76 °C. Actin denaturation had a stronger effect than myosin denaturation on texture changes, whereas myosin denaturation was responsible for changes in color and appearance. A better preservation of tuna quality was obtained by novel sous‐vide cooking over the traditional sous‐vide method.

Practical applications

The results of this study are useful to both the research community and industry because they provide quantitative characterization of the consequences of sous‐vide cooking method on food quality explained by estimating TPD. Moreover, the kinetic parameters of the denaturation rate collected for kinetic modeling of the TPD of tuna, not only have application to simulate denaturation of actin and myosin under different thermal schedules of sous‐vide cooking, but also they can be used for the analysis of additional thermal treatments.     

Influence of tungsten–carbon mixed layer and irradiation defects on deuterium retention behavior in tungsten

The D₂⁺ fluence dependence on deuterium (D) retention was studied to clarify the D retention mechanism in tungsten. The additional D desorption stage was observed around 660 K in the TDS spectrum for a sample implanted with D₂⁺ up to the fluence of 10²³ D⁺ m^?2, which desorption stage was not observed the D₂⁺ implanted sample with the fluence less than 10²² D⁺ m^?2. The TEM observation showed that the highly dense voids were formed in tungsten by D₂⁺ implantation with the fluence of 10²³ D⁺ m^?2, considering that the D would be trapped by voids. To understand the D trapping by voids in C⁺ implanted tungsten, C⁺–D₂⁺ sequential implantation experiments at various C⁺ implantation temperatures were performed. It was found that the amount of D desorbed around 560 K was increased by increasing the C⁺ implantation temperature. The formation of the voids was observed with increasing the C⁺ implantation temperature by TEM, indicating that the increase of D desorption around 560 K was caused by the formation of voids. However, the desorption temperature of D trapped by voids in C⁺ implanted sample was lower than that in D₂⁺ implanted one. TEM observation and XPS measurement indicated that this difference was caused by the increase of void size and/or the presence of implanted carbon.     

Simulation study of the influence of different urban canyons element on the canyon thermal environment

The heat island effect is an important issue for large cities, especially those located in hot and moist climates. The phenomenon is more severe in urban canyons because surrounding high-rise buildings allow little ventilation and dissipation of heat caused by traffic. The primary goal of the present study is to investigate the thermal environment of a major street in Osaka by intensive measurement during the summer of 2006. Osaka is the second largest city in Japan and suffers from the most severe heat island effect. In addition, several fundamental renovations and a composite renovation for the improvement of thermal environment in the urban canyon are proposed, and the efficacies of these measures are verified by computational fluid dynamics (CFD) simulation. It was found that by modifying the heights of buildings along the street and the ground surface materials and increasing the quantity of vegetation, the thermal environment can be improved by a 2.0°C. reduction in standard new effective temperature (SET^*) at maximum.     

Analysis of glycopeptides using lectin affinity chromatography with MALDI-TOF mass spectrometry

Glycopeptides prepared from 1 nmol of a mixture of glycoproteins, transferrin, and ribonuclease B by lysylendopeptidase digestion were isolated by lectin and cellulose column chromatographies, and then they were analyzed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry and MALDI-quadrupole ion trap (QIT)-TOF mass spectrometry which enables the performance of MS ( n ) analysis. The lectin affinity preparation of glycopeptides with Sambucus nigra agglutinin and concanavalin A provides the glycan structure outlines for the sialyl linkage and the core structure of N-glycans. Such structural estimation was confirmed by MALDI-TOF MS and MALDI-QIT-TOF MS/MS. Amino acid sequences and location of glycosylation sites were determined by MALDI-QIT-TOF MS/MS/MS. Taken together, the combination of lectin column chromatography, MALDI-TOF MS, and MALDI-QIT-TOF MS ( n ) provides an easy way for the structural estimation of glycans and the rapid analysis of glycoproteomics.     

Isolation and quantitative detection of tetrachloroethene (PCE)-dechlorinating bacteria in unsaturated subsurface soils contaminated with chloroethenes

The estimation of tetrachloethene (PCE) dechlorinating-activity and identification of PCE-dechlorinating bacteria were performed in 65 unsaturated subsurface soils (at a depth 30-60 cm) that were collected from 21 noncontaminated soils and 44 chloroethene-contaminated soils including four soils that dechlorinated PCE to 1,2-cis-dichloroethene (cisDCE) in situ. Sixteen out of the 44 PCE-contaminated soils and three out of the 21 noncontaminated soils dechlorinated PCE to trichloroethene and cisDCE but not vinyl chloride or ethene after a month of incubation with 0.1% yeast extract at 30 degrees C. Desulfitobacterium sp. strain B31e3 that can dechlorinate PCE to cisDCE was isolated from a soil that dechlorinated PCE to cisDCE in situ. 16S rRNA gene of this strain showed the closest similarity of 99.1% with that of Desulfitobacterium hafniense (formally frappieri) strain DP7. Real-time PCR using specific primer sets targeted to the 16S rRNA genes of the representative PCE-dechlorinating bacteria, Dehalococcoides spp., Desulfitobacterium spp., and Dehalobacter spp. were performed using five unsaturated subsurface soils that dechlorinated PCE and three that did not dechlorinate PCE. In two out of the five soils that dechlorinated PCE, Desulfitobacterium spp. (0.12, 0.38% of total bacteria) and Dehalobacter spp. (0.0045, 0.0061% of total bacteria) were detected, and in one of the five soils, only Desulfitobacterium spp. (0.042% of total bacteria) was detected. None of these representative PCE-dechlorinating bacteria were detected in two out of the five soils that dechlorinated PCE and in all of the three soils that did not dechlorinate PCE. Dehalococcoides spp. were not detected in any unsaturated subsurface soils used in this study. These results suggested the involvement of Desulfitobacterium spp. and probably Dehalobacter spp. rather than Dehalococcoides spp. in the dechlorination of PCE to cisDCE in unsaturated subsurface soils.     

Analysis of pesticides including chlorine in welsh onions and mushrooms using gas chromatograph with an atomic emission detector (GC-AED)

An analytical method for the determination of 32 kinds of pesticide residues in onions, Welsh onions and mushrooms using gas chromatograph with an atomic emission detector (GC-AED) was developed. The pesticides were extracted with acetone-n-hexane (2:3) mixture. The crude extract was partitioned between 5% sodium chloride and ethyl acetate-n-hexane (1:4) mixture. The extract was passed through a Florisil mini-column for cleanup with 10 mL of acetone-n-hexane (1:9) mixture. Although the sensitivity of GC-AED was inferior to that of GC-ECD, GC-AED has a superior element-selectivity. Therefore pesticide residues in foods could be analyzed more exactly by using GC-AED. Thirty-two pesticides including chlorine in onion, Welsh onion and shiitake mushroom were detected without interference. Recoveries of these pesticides from samples determined by GC-AED were 64-114%, except for a few pesticides.     

Analysis of imazalil and its major metabolite in citrus fruits by GC-FTD

A simplified simultaneous analytical method of imazalil (IZ) and its major metabolite, alpha-(2,4-dichlorophenyl)-1H-imidazole-1-ethanol (IZM), in citrus fruits was developed, and commodities samples were investigated. A homogenate of citrus fruits was extracted with ethyl acetate under basic conditions. The crude extract was partitioned between 0.025 mol/L of sulfuric acid and ethyl acetate. The analytes were extracted from the aqueous fraction under basic conditions with ethyl acetate. The extract solution was purified with an ENVI-Carb cartridge, and then analyzed by GC-FTD and GC/MS. Recoveries of IZ and IZM added to grapefruit at the level of 0.05 microgram/g were 90.0 and 108.7%, and those in the case of lemon were 100.4 and 93.0%, respectively. The detection limits were 0.01 microgram/g in samples. By this method, IZ and IZM were analyzed in 46 citrus fruits on the market and were detected simultaneously in some samples.