Comparative proteomic analysis of B. cenocepacia using two-dimensional liquid separations coupled with mass spectrometry

Burkholderia cenocepacia is an important respiratory pathogen in persons with cystic fibrosis. We compared the proteomes of clinical and environmental isolates of B. cenocepacia by using a 2D liquid separation method coupled with mass spectrometry. Proteome maps of four B. cenocepacia isolates were generated. In the first dimension, 5 mg of protein from each isolate was fractionated by chromatofocusing (CF) in the range of pH 4.0-7.0. In the second dimension, each CF fraction was separated by NPS-RP-HPLC. Results of the 2D liquid separation were visualized as 2D UV maps, which allowed direct comparison of proteomes with high resolution and reproducibility. From the proteomic comparison of the four isolates, 38 of 96 differentially abundant proteins were identified by peptide mass fingerprinting and MS/MS sequence analysis using a partially annotated B. cenocepacia protein database. Many of the identified proteins in the clinical isolates are involved in gene translation and bacterial virulence such as transmissibility, resistance, and quorum sensing.     

Comparative proteomic profiling of mammalian cell lysates using phosphopeptide microarrays

A library of 176 human phosphotyrosine-containing peptides was used to establish cell lysate binding profiles in a two colour microarray format. The resulting hits led to the pull-down and identification of biomarkers associated with cancer states.     

A proteomic approach to mimic fibrosis disease evolvement by an in vitro cell line.

Subepithelial fibrosis in asthma involves an increase in the thickening of the lamina reticularis and is due to increased deposition of collagen I, III and V, and fibronectin. The cause of the thickening of the reticular layer is not known in detail, however, it is proposed to be caused by bronchial myofibroblasts. The transformation of fibroblasts to myofibroblasts may be contributed by inflammatory cytokines. In this paper we have studied and compared in vivo tissue material with a human fibroblast target cell. A normal primary fetal fibroblast cell line and HFL-1 (human fibroblast lurg cells) were used as a comparison between fibroblasts from human central biopsies regarding morphology and cell proliferation. Both cell morphology and cell proliferation rate was similar between the different set of cell cultures. Furthermore, it could be concluded that fibroblasts cultures from patients with asthma were surrounded by more extracellular matrix molecules compared to the primary cell line HFL-1, which may mimic the in vivo situation during formation of fibrosis. We wanted to investigate if differential protein display by two-dimensional (2-D) gel electrophoresis and subsequent protein identification by matrix assisted laser desorption/ionization-time of flight (MALDI-TOF)-mass spectrometry could reveal proteins induced by cytokine stimulation that can be correlated to the transformation of normal human fetal lungs cells into a more myofibroblast like phenotype. After stimulation with transforming growth factor-beta (TGF-beta) several myofibroblast markers were found to be regulated. Especially cytoskeletal and cytoskeletal-associated proteins like actin isoforms and tropomyosin, proteins that are responsible for contraction as well as transportation of extra cellular matrix proteins, which are overproduced in the formation of fibrosis. These results indicate that TGF-beta, which is increased in a fibrotic process, participates in the transformation of fibroblasts to myofibroblasts.     

Comparative proteomic analysis of plasma of children with congenital heart disease

Congenital heart disease is one of the largest class of birth defects. Eight subjects with ventricular septal defect (VSD, a kind of congenital heart disease) and 11 health children were enrolled in tandem mass tags label‐based quantitative proteomic analysis to compare plasma proteins differentially abundance. A total of 66 proteins were significantly upregulated or downregulated in VSD patients compared with healthy children. These proteins were involved in pathways linked to platelet activation, fructose and mannose metabolism, complement and coagulation cascades, glycolysis/gluconeogenesis, regulation of actin cytoskeleton, and carbon metabolism. The amount of ten proteins changed significantly (p < 0.05) in newly recruited 30 VSD compared with 15 control children, which were validated by ELISA. The areas under the receiver operating characteristic curve values of fructose‐bisphosphate aldolase B (ALDOB) and thymosin beta‐4 (Tβ4) were higher than those of other candidate proteins. ALDOB and Tβ4 might be potential biomarkers applied for identifying VSD in the further works.     

Comparative analysis of proteomic changes in contrasting flax cultivars upon cadmium exposure

Cadmium (Cd) is classified as a serious pollutant due to its high toxicity, high carcinogenicity, and widespread presence in the environment. Phytoremediation represents an effective low‐cost approach for removing pollutants from contaminated soils, and a crop with significant phytoremediation potential is flax. However, significant differences in Cd accumulation and tolerance were previously found among commercial flax cultivars. Notably, cv. Jitka showed substantially higher tolerance to elevated Cd levels in soil and plant tissues than cv. Tábor. Here, significant changes in the expression of 14 proteins (related to disease/defense, metabolism, protein destination and storage, signal transduction, energy and cell structure) were detected by image and mass spectrometric analysis of two‐dimensionally separated proteins extracted from Cd‐treated cell suspension cultures derived from these contrasting cultivars. Further, two proteins, ferritin and glutamine synthetase (a key enzyme in glutathione biosynthesis), were only up‐regulated by Cd in cv. Jitka, indicating that Cd tolerance mechanisms in this cultivar may include maintenance of low Cd levels at sensitive sites by ferritin and low‐molecular weight thiol peptides binding Cd. The identified changes could facilitate marker‐assisted breeding for Cd tolerance and the development of transgenic flax lines with enhanced Cd tolerance and accumulation capacities for phytoremediating Cd‐contaminated soils.     

Comparative proteomic analysis of round and elongated spermatids during spermiogenesis in mice

Spermiogenesis in mammals is an exclusive process during which haploid round spermatids mature into spermatozoa in the testis. Any abnormality in the process of spermiogenesis may result in male infertility. The aim of the present study was to characterize the differentially expressed proteins between round and elongated spermatids in mice using label-free quantitative mass spectrometry. Of the 2411 proteins identified in this study, 333 were differentially expressed with a ≥10-fold change, including 208 upregulated proteins and 125 downregulated proteins in round spermatids relative to elongated spermatids. Gene Ontology analysis showed that these differentially expressed proteins were categorized into 10 types of subcellular localizations, 9 molecular functions, and were involved in 9 biological processes. All the identified proteins participated in 268 different pathways. In addition, ubiquitin-mediated proteolysis and the proteasome pathway, autophagy, lysosome, and apoptosis pathways were involved in the mechanism of spermiogenesis. Our data may provide valuable information for a better understanding of spermiogenesis and help improve the diagnosis and treatment of male factor infertility.     

Et3B induced stereoselective radical addition of Ph3GeH to acetylenes and its application to isomerization of olefins

Triphenylgermane adds easily to acetylenes in the presence of Et₃B to give (E)- or (Z)-alkenyltriphenylgermanes, respectively, under excellent control of regio- and stereoselectivities.     

Comparative proteomic analysis of grain development in two spring wheat varieties under drought stress

Two spring wheat varieties Ningchun 4 and Chinese Spring with good and poor resistance to abiotic stress, respectively, were used to investigate proteomic changes in the developing grains under drought stress by a comparative proteomics approach. A total of 152 protein spots showed at least twofold differences in abundance on two-dimensional electrophoresis (2-DE) maps, of which 28 and 68 protein spots were identified by MALDI-TOF and MALDI-TOF/TOF mass spectrometry, respectively. Of the 96 identified protein spots, six different expression patterns were found and they were involved in stress/defense/detoxification, carbohydrate metabolism, photosynthesis, nitrogen metabolism, storage proteins and some other important functions. Comparative proteomic analysis revealed that under the drought conditions the decreased degree of ascorbate peroxidases was more significant in Chinese Spring than in Ningchun 4 during grain development whereas translationally controlled tumor protein, which was significantly upregulated at 14 DAF, was present in Ningchun 4 and absent in Chinese Spring. The Rubisco large subunit displayed an upregulated expression pattern in Ningchun 4. In addition, two drought-tolerant proteins, triosephosphate isomerase and oxygen-evolving complex showed B and F type expression patterns in Chinese Spring, but D and B types in Ningchun 4, respectively. These differentially expressed proteins might be responsible for the stronger drought resistance of Ningchun 4 compared to Chinese Spring.     

Comparative gel‐based proteomic analysis of chemically crosslinked complexes in dystrophic skeletal muscle

Duchenne muscular dystrophy is a highly progressive muscle wasting disease with a complex pathophysiology that is based on primary abnormalities in the dystrophin gene. In order to study potential changes in the oligomerization of high‐molecular‐mass protein complexes in dystrophic skeletal muscle, chemical crosslinking was combined with mass spectrometric analysis. The biochemical stabilization of protein interactions was carried out with the homo‐bifunctional and amine‐reactive agent bis[sulfosuccinimidyl]suberate, followed by protein shift analysis in one‐dimensional gels. The proteomic approach identified 11 and 15 protein species in wild type versus dystrophic microsomal fractions, respectively, as well as eight common proteins, with an electrophoretic mobility shift to very high molecular mass following chemical crosslinking. In dystrophin‐deficient preparations, several protein species with an increased tendency of oligomerisation were identified as components of the sarcolemma and its associated intra‐ and extracellular structures, as well as mitochondria. This included the sarcolemmal proteins myoferlin and caveolin, the cytoskeletal components vimentin and tubulin, extracellular collagen alpha‐1(XII) and the mitochondrial trifunctional enzyme and oxoglutarate dehydrogenase. These changes are probably related to structural and metabolic adaptations, especially cellular repair processes, which agrees with the increased oligomerisation of myosin‐3, myosin‐9 and actin, and their role in cellular regeneration and structural adjustments in dystrophinopathy.     

Free flow electrophoresis coupled with liquid chromatography-mass spectrometry for a proteomic study of the human cell line (K562/CR3)

The requirement for prefractionation in proteomic analysis is linked to the challenge of performing such an analysis on complex biological samples and identifying low level components in the presence of numerous abundant housekeeping and structural proteins. The employment of a preliminary fractionation step results in a reduction of complexity in an individual fraction and permits more complete liquid chromatography/mass spectrometry (LC/MS) analysis. Free flow electrophoresis (FFE), a solution-based preparative isoelectric focusing technique, fractionates and enriches protein fractions according to their charge differences and is orthogonal in selectivity to the popular reversed phase high performance liquid chromatography (HPLC) fractionation step. In this paper, we explored the advantages of a combination of FFE and liquid chromatography/mass spectrometry to extend the dynamic range of a proteomic analysis of a complex cell lysate. In this study, the whole cell lysate of a chronic myelogeneous leukemia cell line, K562/CR3, was prefractionated by FFE into 96 fractions spanning pH 3-12. Of these, 35 fractions were digested with trypsin and then analyzed by LC/MS. Depending on the algorithm used for peptide assignment from MS/MS data, at least 319 proteins were identified through database searches. The results also suggested that pI could serve as an additional criterion besides peptide fragmentation pattern for protein identification, although in some cases, a pI shift might indicate post-translational modification. In summary, this study demonstrated that free flow electrophoresis provided a useful prefractionation step for proteomic analysis and when combined with LC/MS allowed the identification of significant number of low level proteins in complex samples.     

14-3-3 protein beta/alpha as a urinary biomarker for renal cell carcinoma: proteomic analysis of cyst fluid

Although various samples, including tissue, cells, serum, and urine, from patients with renal cell carcinoma (RCC) have been analyzed, biomarkers with diagnostic value have yet to be identified. We used a proteomics approach to analyze cyst fluid in cases of cyst-associated RCC to identify accessible and abundant proteins that are overexpressed and/or secreted by RCC cells. Proteins in the cyst fluid were separated by reverse-phase high-performance liquid chromatography and agarose two-dimensional gel electrophoresis and were identified by tandem mass spectrometry. We conducted a National Center for Biotechnology Information search and a MEDLINE search to predict the function of these identified proteins and to select a tumor-marker candidate protein. Our search resulted in the identification and selection of the differentially regulated protein known as 14-3-3 protein beta/alpha, which was overexpressed in cyst fluid from cyst-associated RCC but has not been previously associated with RCC. We then measured its incidence through Western blotting of various normal and RCC samples (serum, urine, tissue, and cyst fluid). The expression levels of 14-3-3 protein beta/alpha were higher in urine samples from patients with RCC than in samples from healthy volunteers. Receiver operating characteristic (ROC) curve analyses were performed to assess this potential biomarker; these data (area under the ROC curve value was 0.8813) indicate a high degree of accuracy for this screening method. 14-3-3 Protein beta/alpha may be a diagnostically useful biomarker for early diagnosis of RCC.     

Photoactivated [3+2] addition of 6,7-seco-angustilobine B to fullerene [C60

A synthesis of a new alkaloid-fullerene conjugate (1) is reported. The reaction was carried out by photoinduced [3+2] cycloaddition of the Alstonia indole alkaloid, 6,7-seco-angustilobine B (2), to fullerene[C60] (3) under aerobic conditions. The major monoaddition photoadduct (1) was characterized unambiguously by UV, IR, MALDI-TOFMS and NMR experiments. A mechanism highlighted by sequential photoinduced electron transfer andradical recombination pathways is also proposed. No significant enhancement in inhibition against M. tuberculosis H37Rv was observed for 1 compared with its parent compounds 2 and 3.     

Synthesis of allene triazole through iron catalyzed regioselective addition to propargyl alcohols

Allene triazole derivatives were successfully synthesized for the first time through iron catalyzed regioselective triazole addition to tertiary propargyl alcohols. The reaction proceeds under mild conditions, giving the desired allene-triazoles in good to excellent yields (up to 96%). The resulting allene-triazoles were confirmed by X-ray crystallography and indicated improved stability.     

Quantitative proteomic analysis of inorganic phosphate-induced murine MC3T3-E1 osteoblast cells

Cleavable isotope-coded affinity tag (cICAT) reagents were utilized to identify and quantitate protein expression differences in control and inorganic phosphate-treated murine MC3T3-E1 osteoblast cells. Proteins extracted from control and treated cells were labeled with the light and heavy isotopic versions of cICAT reagents, respectively. The cICAT-labeled samples were combined, proteolytically digested, and the cICAT-derivatized peptides isolated using immobilized avidin chromatography. The cICAT-labeled peptides were resolved into 96 fractions by strong cation-exchange (SCX) liquid chromatography (LC). Analysis of the SCX-LC cICAT peptide fractions by microcapillary reversed-phase LC-tandem mass spectrometry resulted in the identification and quantitation of 7227 unique peptides corresponding to 2501 proteins, or roughly 9% of the proteins currently predicted to be encoded by the mouse genome. A false positive analysis indicated a 98% confidence in the peptide identifications. To corroborate changes in abundance measured by cICAT with those detectable in traditionally prepared cell lysate, we chose to analyze cyclin D1. Cyclin D1 has been previously identified as a phosphate-responsive gene and was likewise identified as a phosphate-responsive protein in the current analysis. The 1.76-fold increase in abundance in cyclin D1 determined from cICAT corresponds well with the 2.41-fold increase as determined by Western blotting. These results demonstrate that quantitative proteomics is capable of providing a quantitative view of thousands of proteins in mammalian cells within a defined set of experiments.     

三聚磷酸钠与柠檬酸钠钙螯合机理和螯合能力的对比分析

选取了常用洗涤助剂三聚磷酸钠(STPP)与柠檬酸钠(NaCA)为研究体系,通过实验测定和理论分析,对洗涤助剂的钙螯合能力进行了研究.实验研究采用铬黑T指示剂络合滴定法,分别测定以上2种助剂的钙螯合能力,结果表明:2种助剂均可与Ca2+离子形成稳定的螯合物,但螯合Ca2+离子的能力不同,三聚磷酸钠螯合能力较强.理论计算运用Gaussian09程序包,采用密度泛函的B3LYP方法,6-31G(d,p)基组,在溶剂化条件下计算出2种洗涤助剂与Ca2+离子形成螯合物的几何结构及其结合能,三聚磷酸钠和柠檬酸钠与Ca2+离子的螯合物均为六配位八面体结构,磷酸基团或羧酸基团与羟基参与配位.实验所得螯合能力的差异可以由理论计算得到的分子结构、结合能及前线分子轨道方面进行解释.     

Selective inhibition of the radical addition of thiols to olefins by iron pentacarbonyl

Conclusions The effect of Fe(CO)₅ and [Fe(CO)₃SC₂H₅]₂ on the competitive recombination of thiyl radicals and their addition to olefins was studied. Iron carbonyls selectively inhibit the addition and catalyze the recombination.Translated from Izvestiya Akademii Nauk SSSR, Seriya Khimicheskaya, No. 7, pp. 1647–1649, July, 1972.     

The addition of butanethiol to butyl propiolate in the presence of iron carbonyls

Conclusions The reactions of thiols with electrophilic compounds, catalyzed by ironpentacarbonyl and hexacarbonyldiethylthiodiiron, have a nucleophilic addition mechanism.Translated from Izvestiya Akademii Nauk SSSR, Seriya Khimicheskaya, No. 10, pp. 2222–2224, October, 1970.