Resolution and characterisation of multiple isoforms of bovine kappa-casein by 2-DE following a reversible cysteine-tagging enrichment strategy

Visualisation of multiple isoforms of kappa-casein on 2-D gels is restricted by the abundant alpha- and beta-caseins that not only limit gel loading but also migrate to similar regions as the more acidic kappa-casein isoforms. To overcome this problem, we took advantage of the absence of cysteine residues in alpha(S1)- and beta-casein by devising an affinity enrichment procedure based on reversible biotinylation of cysteine residues. Affinity capture of cysteine-containing proteins on avidin allowed the removal of the vast majority of alpha(S1)- and beta-casein, and on subsequent 2-D gel analysis 16 gel spots were identified as kappa-casein by PMF. Further analysis of the C-terminal tryptic peptide along with structural predictions based on mobility on the 2-D gel allowed us to assign identities to each spot in terms of genetic variant (A or B), phosphorylation status (1, 2 or 3) and glycosylation status (from 0 to 6). Eight isoforms of the A and B variants with the same PTMs were observed. When the casein fraction of milk from a single cow, homozygous for the B variant of kappa-casein, was used as the starting material, 17 isoforms from 13 gel spots were characterised. Analysis of isoforms of low abundance proved challenging due to the low amount of material that could be extracted from the gels as well as the lability of the PTMs during MS analysis. However, we were able to identify a previously unrecognised site, T(166), that could be phosphorylated or glycosylated. Despite many decades of analysis of milk proteins, the reasons for this high level of heterogeneity are still not clear.     

Molecular imprinted polymer with cloned bacterial protein template enriches authentic target in cell extract

Here we describe a new method for preparing a protein-imprinted polymer with a cloned bacterial protein template, which recognizes/adsorbs authentic target protein present at a relatively low level in cell extract. In this work, cloned pig cyclophilin 18 (pCyP18) was used as a template. The template protein was selectively assembled with memory molecules from their library, which consists of numerous limited length polymer chains with randomly distributed recognition sites and immobilizing sites. These assemblies of protein and memory molecules were adsorbed by porous polymeric beads and immobilized by cross-linking polymerization. After removing the template, binding sites that were complementary to the target protein in size, shape and the position of recognition groups were exposed, and their confirmation was preserved by the cross-linked structure. The synthesized imprinted polymer was used to adsorb authentic pCyP18 from cell extract, and its proportional content was enriched 300 times.     

From purification of large amounts of phospho-compounds (nucleotides) to enrichment of phospho-peptides using anion-exchanging resin

Through years of practice, mass spectrometry has proven to be one of the most reliable and sensitive methods for the localization of protein phosphorylation sites. Among numerous innovative methods, affinity enrichment such as immobilized metal-ion affinity chromatography followed by liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis appears to be the most widely chosen procedure. Here, I report a method that was originally designed for purification of large amounts of nucleotides using anion-exchanging resin but has shown the promise of enriching phosphorylated peptides. Mixtures composed of uridine monophosphate, uridine diphosphate, uridine triphosphate, and their nonphosphate compound-uridine were bottom-line separated on an anion-exchanging solid-phase extraction (SPE) column by four steps of elution with a gradient of salt concentration and pH values. The miniature form of this SPE column showed significant separation (or enrichment) of the tryptic phospho-peptides from non-phospho-peptides of the standard protein beta-casein with two steps of elution (100mM NaCl and 5% NH(4)OH). Furthermore, after utilization of this anion-exchanging-column enrichment followed by LC/MS/MS analysis on a quadrupole-tine of flight instrument, a new phosphorylation site (S191) in bovine chromogranin A was identified.     

重金属在鲫鱼卵巢的富集及对卵细胞的损伤效应

近些年来,随着国民经济的快速发展,环境和生态问题也日益凸显,其中水域环境受重金属的污染亦愈演愈烈,水生动物的生存受到严峻的挑战。在水环境中,各种重金属元素不但在水生动物的皮肤、肌肉、鳃和其他内脏器官中富集,而且在其赖以生息繁衍、绵延种群的     

A new approach for on-line enrichment in electrophoresis of dilute protein solutions

A method is described for on-line enrichment/zone sharpening of a sample of negatively charged proteins (an analogous method for cationic proteins can be designed). The sample is applied on the top of a 5-mm thick layer of a neutral polyacrylamide gel which rests on another 5-mm thick, large-pore polyacrylamide gel which contains positively charged groups. The latter gel layer is attached to the neutral gel column, used for the electrophoretic separation of the proteins. When a voltage is applied the proteins start migrating and become electrostatically adsorbed at the top of the charged, large-pore gel layer (pH 5.4). With the upper electrode vessel filled with a buffer of a pH higher (pH 7.7) than that employed in the enrichment step and with a voltage between the electrodes, these enriched proteins are released (because the enrichment gel is non-charged at pH 7.7) with zone sharpening and migrate into the 5-cm long column (i.d. 5 mm) of a neutral, large-pore polyacrylamide gel for electrophoretic analysis. Upon the electrophoretic migration from the enrichment gel into the separation gel a second zone sharpening may occur, if the increase in pH from 5.4 to 7.7 in the separation gel is not close to momentary. By employing colored test proteins the efficiency of the enrichment step is visually illustrated by a picture. The principle of the concentration method described has been employed also in chromatographic experiments and can with appropriate modifications also be used in other electrophoretic methods, such as capillary electrophoresis.     

华氏巨球蛋白血症相关突变MYD88 L265P新型检测体系的建立

华氏巨球蛋白血症(Waldenstr9m’s macroglobulinemia,WM)是一种罕见的,不可治愈的淋巴浆细胞淋巴瘤(lymphoplasmacytic lymphoma,LPL)。MYD88 L265P突变在华氏巨球蛋白血症患者中检出率很高(90%),可以用于疾病的鉴别和诊断,因此,开发一种高灵敏度的检测方法对这个突变进行检测具有较大的临床价值。通过将ARMS技术与Clamping PCR技术相结合,建立的新型MYD88 L265P突变富集检测体系可以满足这一需求。优化后,该检测体系检出限为102拷贝,灵敏度为0.1%,对19份临床样品的双盲试验中,检测结果准确率达到100%。所建立的方法具有灵敏、准确的优势,适用于华氏巨球蛋白血症的早期诊断,有较为广阔的应用前景。     

Plant protein phosphorylation monitored by capillary liquid chromatography--element mass spectrometry

Many essential cellular functions such as growth rate, motility, and metabolic activity are linked to reversible protein phosphorylation, since they are controlled by signaling cascades based mainly on phosphorylation/dephosphorylation events. Quantification of global or site-specific protein phosphorylation is not straightforward with standard proteomic techniques. The coupling of capillary liquid chromatography (microLC) with ICP-MS (inductively coupled plasma-mass spectrometry) is a method which allows a quantitative screening of protein extracts for their phosphorus and sulfur content, and thus provides access to the protein phosphorylation degree. In extension of a recent pilot study, we analyzed protein extracts from the model organisms Arabidopsis thaliana and Chlamydomonas reinhardtii as representatives for multicellular and unicellular green photosynthetically active organisms. The results indicate that the average protein phosphorylation level of the algae C. reinhardtii is higher than that of A. thaliana. Both the average phosphorylation levels were found to be between the extreme values determined so far for prokaryotes (C. glutamicum, lowest levels) and eukaryotes (Mus musculus, highest levels). Tissue samples of A. thaliana representing different stages of plant development showed varying levels of protein phosphorylation indicating a different adjustment of the kinase/phosphatase system. We also utilized the microLC-ICP-MS technology to estimate the efficiency of a novel phosphoprotein enrichment method based on aluminum hydroxide, since the enrichment of phosphorylated species is often an essential step for their molecular characterization.     

Cortisol and behavioral responses to enrichment in military working dogs

Our objective was to compare the effects of two schedules of enrichment exposure, a distributed (high frequency) and regular regime versus a mass (low frequency) and irregular regime, on the cortisol and behavioral responses of 14 kennelled military working dogs. The enrichment experiences were exercise coupled with human contact. Both groups of dogs were exposed to approximately the same total amount of enrichment per week for a total of 7 weeks. The distributed and regular regime (DRR) furnished enrichment for 20-min intervals three to four times a day every weekday; the mass and irregular regime (MIR) randomly furnished enrichment for 16 or 24 continuous hours 1 day out of 5. Our data show that: (1) cortisol concentrations of DRR dogs significantly decreased between the first and the last week of observations; this was not the case in MIR dogs; (2) at the 7th week DRR dogs had lower cortisol concentrations than did MIR dogs; (3) all dogs in each group exhibited stereotypies. Our results suggest that MIR dogs underwent a decline in welfare throughout the experiment, whereas DRR dogs appeared to undergo a period of acute stress at the beginning of their training that resolved over 7 weeks. This study suggests that the welfare of kennelled dogs, particularly military working dogs, is affected among other factors by schedules of exercise and contact with people.