水稻孢子体雄性不育系小孢子发育过程中的蛋白质分析

以孢子体雄性不育系马协Ａ、马协Ｂ、马协６３（Ｆ１）和珍汕９７Ａ、珍汕９７Ｂ、汕优６３（Ｆ１）为材料，对小孢子发育的花粉母细胞时期、四分体时期、单核期、二核期、三核期的花药蛋白质进行ＳＤＳ－ＰＡＧＥ分析．结果表明：在马协型细胞质雄性不育系中，有１２种多肽的表达具有时序特征，其中９种多肽可能与马协Ａ雄性不育有关；在野败型细胞质雄性不育系中，有１０种多肽的表达具有时序特征，其中６种多肽可能与珍汕９７Ａ雄性不育有关     

A simple method to remove contaminating salt from IPG strips prior to IEF

In this study, Saccharomyces cerevisiae yeast lysate is used to demonstrate how a simple wash procedure can improve IEF of IPG strips passively rehydrated in the presence of NaCl. By performing three 10 min washes after IPG strip rehydration and before IEF, corresponding second-dimensional gels from strips containing NaCl look similar to control strips while the second-dimensional gels of unwashed strips contains streaks and spaces devoid of protein. Up to 500 mM NaCl was added to the yeast lysate and successfully focused following this wash regime. Protein loss due to the washes was determined to be minimal by comparing replicates of washed and unwashed strips and analyzing the densities of their corresponding second-dimensional gel spots. In the event of unknown salt contamination, indicated by low voltage during focusing, it is possible to stop focusing, wash the strips, and then continue focusing with acceptable second dimension results.     

Property and Application of BACy‐Based Functional Hydrogels

Conventional polyacrylamide hydrogels prepared from the free radical polymerization between acrylamide and N,N′‐methylenebisacrylamide (NMBA) have been frequently used in the biochemical technique like the sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) to resolve protein mixtures. In this study, we have prepared an alternative polyacrylamide hydrogel from the cross‐linking of acrylamide and N,N′‐bisacrylylcystamine (BACy). In addition, we have compared the BACy‐based hydrogel with the NMBA‐based polyacrylamide hydrogel for their physical properties such as swelling ratio, shear modulus, crosslink density and morphology. Moreover, we further determined whether BACy‐based polyacrylamide hydrogel could be applied to SDS‐PAGE and proteomics research. The results showed that this type of hydrogel is capable of separating proteins and facilitates further in‐gel protein digestion and the following protein identifications by mass spectrometry. In summary, our study provides a basis for the putative application of BACy‐based hydrogels.     

Quantitative assessment of multi‐laboratory reproducibility of SDS‐PAGE assays: Digestion pattern of β‐casein and β‐lactoglobulin under simulated conditions

A digestion protocol was applied in triplicate by ten laboratories, simulating in vivo gastric and duodenal conditions. The intra‐ and inter‐laboratory variability in the kinetics of protein degradation was quantified, focussing on the digestion of β‐casein under gastric conditions, and of β‐lactoglobulin (β‐Lg) under duodenal conditions. The addition of surfactants such as phosphatidylcholine (PC) in the digestion mix was also evaluated. Identification and quantification of peptide bands on SDS‐PAGE gels formed the basis for analysis. An average intensity loss of 69% (SD=13.5) at 5 min (89% at 10 min, with SD=5.5) was observed for β‐casein, whereas the β‐Lg duodenal digestion showed an 82% loss at 30 min (SD=14.2). Constant rates of first‐order reactions showed that for fast reactions, inaccuracies in the time of first sampling contributed to the variability, which were also affected by image quality, saturation, and the splitting of time courses across gels. Breakdown products for β‐casein included ten other polypeptides, with four detected in all and two in most gels, and for β‐Lg ten polypeptides, with five detected in most, and two in two‐third of the cases. Addition of PC in the gastric phase led to β‐Lg intensity loss only a quarter as large as without PC and altered β‐Lg proteolysis in the duodenal compartment.     

In situ demonstration and quantitative analysis of the intrinsic properties of glycoside hydrolases

Based on digital image analysis techniques and a series of optimizations in native electrophoresis, a new direct method to simultaneously detect the intrinsic properties of each active component in the enzymatic system of glycoside hydrolase was established. The key technique is that the concentration changes of substrate (or product) on the gel can be determined quantitatively by the gray value changes of the corresponding band after electrophoretic separation. In this manner, the catalytic characteristics of each glycoside hydrolase component were demonstrated in situ and were easily determined after immersing the gel in a series of solutions containing substrates or their derivatives. Because of its high throughput, great sensitivity, and convenient operation, this method can be used to demonstrate the natural diversity of glycoside hydrolases and to study spatial and temporal regulation in multienzyme expression systems. Thus, it is an effective approach to study the functional proteomics of glycoside hydrolases.     

Analysis of oligomeric transition of silkworm small heat shock protein sHSP20.8 using high hydrostatic pressure native PAGE

The small heat shock proteins (sHSPs) solubilize thermo-denatured proteins without adenosine triphosphate energy consumption to facilitate protein refolding. sHSP20.8 is one of the silkworm (Bombyx mori) sHSPs having only one cystein in the N-terminal domain: Cys43. We report a simple measurement of oligomeric transition of sHSP20.8 using high hydrostatic pressure native polyacrylamide gel electrophoresis (high hydrostatic pressure (HP) native polyacrylamide gel electrophoresis (PAGE)). At ambient pressure under oxydative condition, the native PAGE of thermal transition of sHSP20.8 oligomer displayed a cooperative association. In contrast, HP native PAGE clearly demonstrated that sHSP20.8 dissociated at 80 MPa and 25°C, and the resultant molecular species gradually reassociated with time under that condition. In addition, the reassociation process was suppressed in the presence of the reductant. These results are consistent with the idea that sHSP20.8 oligomer temporally dissociates at the first thermo-sensing step and reassociates with the oxidation of Cys43.     

SDS‐PAGE procedure: Application for characterization of new entirely uncharged nucleic acids analogs

SDS‐PAGE is considered to be a universal method for size‐based separation and analysis of proteins. In this study, we applied the principle of SDS‐PAGE to the analysis of new entirely uncharged nucleic acid (NA) analogues, – phosphoryl guanidine oligonucleotides (PGOs). The procedure was also shown to be suitable for morpholino oligonucleotides (PMOs) and peptide nucleic acids (PNAs). It was demonstrated that SDS can establish hydrophobic interactions with these types of synthetic NAs, giving them a net negative charge and thus making these molecules mobile in polyacrylamide slab gels under the influence of an electric field.     

Speciation of aluminium in tea infusions by use of SEC and FPLC with ICP–OES and ES–MS–MS detection

Speciation of Al in tea infusions was studied by size exclusion chromatography (SEC) and anion-exchange fast protein liquid chromatography (FPLC). Fractions were collected throughout the chromatographic separations and Al was determined “off line” by inductively coupled plasma optical emission spectroscopy (ICP–OES). Black, green, and red tea samples were investigated. The total concentration of Al in tea infusions was determined by ICP–OES and ranged between 0.5 and 4 mg dm⁻³. The pH of tea infusions ranged between 5.3 and 5.5. Data from SEC–ICP–OES analysis indicated that 10–35% of total Al in tea infusions was eluted at a retention volume corresponding to a molecular mass of approximately 3800 Da. The remaining Al was adsorbed on the column resin. The same tea infusions were also analysed by anion-exchange FPLC–ICP–OES. It was found experimentally that the same percentage of total Al as from the SEC column was eluted at a retention volume that corresponded to negatively charged Al-citrate. The remaining Al was adsorbed on the column resin. Identification of Al-binding ligands eluting under the chromatographic peak was performed by electrospray ionisation tandem mass spectrometry (ES–MS–MS) analysis. It was proven that ionic Al species in tea infusions (10–35% of the total Al) corresponded to negatively charged Al-citrate. The remaining species that was adsorbed on the SEC or FPLC columns was most probably bound to phenolic compounds. Speciation of Al in tea with milk or lemon was also studied. Results for tea with milk indicated that Al-citrate was not transformed and that approximately 60% of total Al was transformed into high-molecular-mass Al species. This fraction was subjected to sodium dodecyl sulfonate polyacryl gel electrophoresis (SDS–PAGE). The results indicated that Al was occluded by milk proteins (mostly caseins). When citric acid was added to tea infusions the percentage of negatively charged Al-citrate remained either the same or increased to 40% of total Al.     

Differential staining of Western blots of human secreted glycoproteins from serum, milk, saliva, and seminal fluid using lectins displaying diverse sugar specificities

Human milk, serum, saliva, and seminal fluid glycoproteins (gps) nourish and protect newborn and adult tissues. Their saccharides, which resemble cell membrane components, may block pathogen adhesion and infection. In the present study, they were examined by a battery of lectins from plants, animals, and bacteria, using hemagglutination inhibition and Western blot analyses. The lectins included galactophilic ones from Aplysia gonad, Erythrina corallodendron, Maclura pomifera (MPL), peanut, and Pseudomonas aeruginosa (PA-IL); fucose-binding lectins from Pseudomonas aeruginosa (PA-IIL), Ralstonia solanacearum (RSL), and Ulex europaeus (UEA-I), and mannose/glucose-binding Con A. The results demonstrated the chosen lectin efficiency for differential analysis of human secreted gps as compared to CBB staining. They unveiled the diversity of these body fluid gp glycans (those of the milk and seminal fluid being highest): the milk gps interacted most strongly with PA-IIL, followed by RSL; the saliva gps with RSL, followed by PA-IIL and MPL; the serum gps with Con A and MPL, followed by PA-IIL and RSL, and the seminal plasma gps with RSL and MPL, followed by UEA-I and PA-IIL. The potential usage of these lectins as probes for scientific, industrial, and medical purposes, and for quality control of the desired gps is clearly indicated.     

Comparison of the Electrochemical Behavior of the High Molecular Mass Cadmium Proteins in Arabidopsis thaliana and in Vegetable Plants on Using Preparative Native Continuous Polyacrylamide Gel Electrophoresis (PNC‐PAGE)

In Arabidopsis cytosol (supernatant) and in supernatants of vegetable plants high molecular mass cadmium proteins with molecular mass 200 kDa were isolated by using p reparative n ative c ontinuous p olya crylamide g el e lectrophoresis (PNC‐PAGE). Because of a different electrochemical behavior of the Cd proteins in Arabidopsis and endive supernatants on using the same PAGE method, it is concluded that the high molecular mass cadmium proteins of Arabidopsis and endive possess different isoelectric points. Consequently, different chemical structures of the Cd proteins with molecular mass 200 kDa are present in Arabidopsis thaliana and in endive. During the electrophoretic separation of vegetable metalloproteins by using the Model 491 Prep Cell from BioRad, electroanalytical processes like electrode reactions may play an important role.