Proteome analysis of round-headed and normal spermatozoa by 2-D fluorescence difference gel electrophoresis and mass spectrometry

Globozoospermia is a severe form of teratozoospermia characterized by round-headed spermatozoa with an absent acrosome, an aberrant nuclear membrane and midpiece defects. Globozoospermia is diagnosed by the presence of 100% round-headed spermatozoa on semen analysis, and patients with this condition are absolutely infertile. The objective of this study was to investigate the differences in protein expression between human round- headed and normal spermatozoa. Two-dimensional （2-D） fluorescence difference gel electrophoresis （DIGE） coupled with mass spectrometry （MS） was used in this study. Over 61 protein spots were analysed in each paired normal/round-headed comparison, using DIGE technology along with an internal standard. In total, 35 protein spots identified by tandem mass spectrometry （MS/MS） exhibited significant changes （paired t-test, P 〈 0.05） in the expression level between normal and round-headed spermatozoa. A total of nine proteins were found to be upregulated and 26 proteins were found to be downregulated in round-headed spermatozoa compared with normal spermatozoa. The differentially expressed proteins that we identified may have important roles in a variety of cellular processes and structures, including spermatogenesis, cell skeleton, metabolism and spermatozoa motility.

Establishment of a high-resolution 2-D reference map of human spermatozoal proteins from 12 fertile sperm-bank donors

Aim： To extend the analysis of the proteome of human spermatozoa and establish a 2-D gel electrophoresis （2-DE） reference map of human spermatozoal proteins in a pH range of 3.5-9.0. Methods： In order to reveal more protein spots, immobilized pH gradient strips （24 cm） of broad range of pH 3-10 and the narrower range of pH 6-9, as well as different overlapping narrow range pH immobilized pH gradient （IPG） strips, including 3.5-4.5, 4.0-5.0, 4.5-5.5, 5.0-6.0 and 5.5-6.7, were used. After 2-DE, several visually identical spots between the different pH range 2-D gel pairs were cut from the gels and confirmed by mass spectrometry and used as landmarks for computer analysis. Results： The 2-D reference map with pH value from 3.5 to 9.0 was synthesized by using the ImageMaster analysis software. The overlapping spots were excluded, so that every spot was counted only once. A total of 3 872 different protein spots were identified from the reference map, an approximately 3-fold increase compared to the broad range pH 3-10 IPG strip （1 306 spots）. Conclusion： The present 2-D pattern is a high resolution 2-D reference map for human fertile spermatozoal protein spots. A comprehensive knowledge of the protein composition of human spermatozoa is very meaningful in studying dysregulation of male fertility.

Identification of differentially expressed proteins in poplar leaves in-duced by Marssonina brunnea f. sp. Multigermtubi

Black spot disease in poplar is a disease of the leaf caused by fungus. The major pathogen is Marssonina brunnea f. sp. multigermtubi. To date, little is known about the molecular mechanism of poplar （M. brunnea） interaction. In order to identify the proteins related to disease resistance and understand its molecular basis, the clone ＂NL895＂ （P. euramericana CL＂NL895＂）, which is highly resistant to M. brunnea f. sp. multigermtubi, was used in this study. We used two-dimensional gel electrophoresis （2-DE） and mass spectrometry （MS） to identify the proteins in poplar leaves that were differentially expressed in response to black spot disease pathogen, M. brunnea f. sp. multigermtubi. Proteins extracted from poplar leaves at 0, 12, 24, 48, and 72 h after pathogen-inoculation were separated by 2-DE, About 500 reproducible protein spots were detected, of which 40 protein spots displayed differential expression in levels and were subjected to Matrix assisted laser desorption/ionization time of flight mass spectrometry （MALDI-TOF MS） followed by database searching. According to the function, the identified proteins were sorted into five categories, that is, protein synthesis, metabolism, defense response and unclassified proteins.

Combining Phytate/Ca^(2+) Fractionation with Trichloroacetic Acid/Acetone Precipitation Improved Separation of Low-Abundant Proteins of Wheat (Triticum aestivum L.) Leaf for Proteomic Analysis

Proteomic assessment of low-abundance leaf proteins is hindered by the large quantity of ribulose-1,5-bisphosphate carboxylase/oxygenase （Rubisco） present within plant leaf tissues. In the present study, total proteins were extracted from wheat （Triticum aestivum L.） leaves by a conventional trichloroacetic acid （TCA）/acetone method and a protocol first developed in this work. Phytate/Ca2＋ fractionation and TCA/acetone precipitation were combined to design an improved TCA/acetone method. The extracted proteins were analysed by two-dimensional gel electrophoresis （2-DE）. The resulting 2-DE images were compared to reveal major differences. The results showed that large quantities of Rubisco were deleted from wheat leaf proteins prepared by the improved method. As many as （758±4） protein spots were detected from 2-DE images of protein extracts obtained by the improved method, 130 more than those detected by the TCA/acetone method. Further analysis indicated that more protein spots could be detected at regions of pI 4.00-4.99 and 6.50-7.00 in the improved method-based 2-DE images. Our findings indicated that the improved method is an efficient protein preparation protocol for separating low-abundance proteins in wheat leaf tissues by 2-DE analysis. The proposed protocol is simple, fast, inexpensive and also applicable to protein preparations of other plants.

CHANGES OF NUCLEAR MATRIX PROTEIN AND ITS RELATIONSHIP WITH c-erbB-2 IN HUMAN COLON ADENOCARCINOMA

Objective： Nuclear matrix protein is tissue, cell-type specific, and tumor-relative. It plays an important role in the regulation of intranuclear processes. Some researches also showed that a c-erbB-2 promoter-specific DNA-binding nuclear matrix protein is present only in malignant human breast tissues and induces mitogenesis and cell surface expression of the c-erbB-2 protein in resting NIH/3T3 cells. But it is not clear that how it in colon adenocarcinomas. Methods： Two-dimensional gel electrophoretic method was used for NMP identification and immunohistochemistry was used for c-erbB-2 detection in 12 cases of colon adenocarcinomas and matched adjacent normal colon tissues. Results： 5 different nuclear matrix proteins （named C1-C5） were identified in 12 colon adenocarcinoma specimens, but not in the matched adjacent normal colon tissues; 3 nuclear matrix proteins （named N1-N3） were identified in all 12 matched adjacent normal colon tissues, but not in colon adenocarcinoma specimens. A nuclear matrix protein （named N4） was detected in all of 9 moderated-well differentiated adenocarcinomas and all 12 matched adjacent normal colon tissues, but not in 3 poor-differentiated adenocarcinomas. All of the 10 colon adenocarcinomas which had the nuclear matrix protein C4 were c-erbB-2 expression positive. Conclusion： The data suggest that there are specific nuclear matrix proteins in colon adenocarcinomas and its subtypes, which maybe valuable to serve as markers of colon adenocarcinomas in future. Nuclear matrix protein C4 probably is a c-erbB-2 promotor-specific nuclear matrix protein in colon adenocarcinomas, and may induce the expression of c-erbB-2.

Plant Proteomics in the Post-genomic Era

Proteomics is one of the most active research fields in the post-genomic era. Here we briefly introduce the scientific background of the origination of proteomics and its content, research method. The new developments of proteomics at the levels of individual plants, tissues, organs and organells, as well as its applications in the area of plant genetic diversity, mutant characterization, and plant physiology, etc are reviewed. At last, the challenge and prospect of proteomics are discussed.

5-Aminolevulinic acid alleviates herbicide-induced physiological and ultrastructural changes in Brassica napus

It is well known that application of 5-aminolevuJinic acid （ALA） could promote the plant growth under abiotic stress in oilseed rape （Brassica napus L.）. However, the specifics of its physiological and ultrastructural regulation under herbi- cide stress conditions are poorly understood. In the present study, alleviating role of ALA in B. napus was investigated under four levels of herbicide propyl 4-（2-（4,6-dimethoxypyrimidin-2-yloxy） benzylamino） benzoate （ZJ0273） （0, 100, 200 and 500 mg L-1） with or without 1 mg L-1 ALA treated for 48 or 72 h. Results showed that after 48 h of herbicide stress, the growth of rape seedlings was significantly inhibited with the successive increases of the ZJ0273 concentrations from 0 to 500 mg L-1, but this inhibition was obviously alleviated by exogenous application of ALA. However, when treatment time prolonged to 72 h, the recovery effects of ALA could not be evaluated due to the death of plants treated with the highest concentration of ZJ0273 （500 mg L-1）. Further, the root oxidizability and activities of antioxidant enzymes （superoxide dismutase, perox- idase and ascorbate peroxidase） were dramatically enhanced by the application of 1 mg L-1 ALA under herbicide stress. Therefore, plants treated with ALA dynamically modulated their antioxidant defenses to reduce reactive oxygen species （ROS） accumulation and malondialdehyde （MDA） content induced by herbicide stress. Additionally, exogenously applied ALA improved the ultrastructure＇s of chloroplast, mitochondria and nucleus, and induced the production of stress proteins. Our results suggest that ALA could be considered as a potential plant growth regulator for the improvement of herbicide tolerance through alleviation of the physiological and ultrastructural changes induced by the herbicide in crop production.

Application of proteome technology in screening biomarkers associated with gastric cancer

Objective: To initially explore the application of proteome technologies in study of serum, to establish two-dimen-sional gel electrophoresis (2-DE) profiles of human gastric cancer serum and paired normal serum, and to screen and identify differentially expressed proteins in poorly-differentiated gastric cancer serum and paired normal serum, in which try to find out significant biomarker candidates for gastric cancer. Methods: 2-DE was adopted to separate the total proteins of poorly-differentiated gastric cancer serum and paired normal serum. After staining and analyzing by ImageMaster 2D Elite software, the differentially expressed proteins were identified by matrix-assisted laser desorption/ionization-time of flight- mass spectrometry (MALDI-TOF-MS). Results: 2-DE serum profiles with high resolution were obtained. Five protein spots were found as differentially-expressed proteins and identified as Serpin B6 (Placental thrombin inhibitor) Cytoplasmic antiproteinase (CAP) (Protease inhibitor6) (PI-6), Septin-1 (LARP) (Serologically defined breast cancer antigen NY-BR-24), Kallikrein-6 precursor (Protease M) (Neurosin) (Zyme) (SP59), Hemoglobin beta chain, Hemoglobin beta chain and Beta-defensin 108 precursor (Beta-defensin 8) (DEFB-8). Conclusion: The differential proteins were demonstrated to present in poorly-differentiated gastric cancer serum and paired normal serum. The molecular biomarkers associated with poorly differentiated gastric cancer could be possibly identified by the high throughput screening proteome technology.

Effect of high salinity on cell growth and protein production of Antarctic ice microalgae Chlamydomonas sp. ICE-L

Antarctic ice microalgae Chlamydomonas sp.ICE-L can survive and thrive in Antarctic sea ice.In this study,Chlamydomonas sp.ICE-L could survive at the salinity of 132‰ NaCl.SDS-PAGE showed that the density of 2 bands（26 and 36 kD） decreased obviously at the salinity of 99‰ NaCl compared to at the salinity of 33‰ NaCl.The soluble proteins in Chlamydomonas sp.ICE-L grown under salinity of 33‰ and 99% NaCl were compared by 2-D gel electrophoresis.After shocking with high salinity,8 protein spots were found to disappear,and the density of 28 protein spots decreased.In addition,19 protein spots were enhanced or induced,including one new peptide（51 kD）.The changes of proteins might be correlated with the resistance for Chlamydomonas sp.ICE-L to high salinity.

Increased RhoGDI2 and peroxiredoxin 5 levels in asthmatic murine model of β2-adrenoceptor desensitization： A proteomics approach

Background β2-adrenoceptor （β2AR） desensitization is a common problem in clinical practice, β2AR desensitization proceeds by at least such three mechanisms as heterologous desensitization, homologous desensitization and a kind of agonist-induced rapid phosphorylation by a variety of serine/threonine kinases. It is not clear whether there are other mechanisms, This study aimed to investigate potential mechanisms of β2AR desensitization.Methods Twenty-four BALB/c （6-8 weeks old） mice were divided into three groups, which is, group A, phosphate buffered saline （PBS）-treated; group B, ovalbumin （OVA）-induced; and group C, salbutamol-treated. Inflammatory cell counts, cytokine concentrations of bronchoalveolar lavage fluid （BALF）, pathological sections, total serum IgE, airway responsiveness, membrane receptor numbers and total amount of β2AR were observed. Asthmatic mouse model and β2AR desensitization asthmatic mouse model were established. Groups B and C were selected for two-dimensional gel electrophoresis （2DE） analysis so as to find key protein spots related to β2AR desensitization.Results Asthmatic mouse model and β2AR desensitization asthmatic mouse model were verified by inflammatory cell count, cytokine concentration of BALF, serum IgE level, airway hyperreactivity measurement, radioligand receptor binding assay, Western blot analysis, and pathologic examination. Then the two groups （groups B and C） were subjected to 2DE. Two key protein spots associated with β2AR desensitization, Rho GDP-dissociation inhibitor 2 （RhoGDl2） and peroxiredoxin 5, were found by comparative proteomics （2DE and mass spectrum analysis）.Conclusion Oxidative stress and small G protein regulators may play an important role in the process of β2AR desensitization.v

Proteomics analysis of cerebral cortex in Wistar rats

To analyze the protein expression pattern of the cerebral cortex in Wistar rats using the proteomics approach,proteins were separated by two-dimensional gel electrophoresis,stained with Coomassie brilliant blue and digested with trypsin.Then,we analyzed the peptide section using a matrix-assisted laser desorption/ionization time of flight mass spectrometry(MALDI-TOF-MS)and identified the protein by indexing special database(SwissProt)according to the finger printing of the peptide quality.Eighty-four protein spots were identified,including metabolic enzymes,skeleton proteins,heat shock proteins,antioxidant proteins,signaling proteins,proteasome related proteins,neuron and glial specific proteins and serum associated proteins.The result of this study enriches the database of the proteome in the cerebral cortex of rats and lays a foundation for further research of neurological disorders in rat models.