Search for peptide sequences involved in human antisperm antibody-mediated male immunoinfertility by using phage display technology

The aim of the present study was to delineate peptide sequences against which antisperm antibodies (ASA) are raised in immunoinfertile men. Using the phage display technology, seven unique and novel dodecamer amino acid sequences were identified that reacted with the sera of immunoinfertile men. The peptides were synthesized based upon these amino acid sequences and examined for their immunoreactivity with sera from ASA-positive immunofertile men (n = 15) and ASA-negative fertile men (n = 18) for IgM, IgG, and IgA class of antibody in the enzyme-linked immunosorbent assay (ELISA). All the seven synthetic peptides showed a significantly (P < 0.001) higher mean absorbance values for IgG and/or IgA class of antibody with the immunoinfertile sera compared to fertile control sera. Three of the seven peptides demonstrated a stronger reaction (>2 SD units) with 27%-40% of immunoinfertile sera compared to fertile controls. These peptide sequences may find applications in the specific diagnosis and treatment of immunoinfertility and in contraceptive vaccine development. The phage display technique provides an exciting and novel technology to delineate sperm epitopes involved in immunoinfertility.     

Human sperm proteome: immunodominant sperm surface antigens identified with sera from infertile men and women.

The objective of this study was to identify those immunodominant sperm antigens recognized by antisperm antibodies (ASA) in the serum samples of infertile men and women. High-resolution two-dimensional gel electrophoresis was employed to separate human sperm proteins using isoelectric focusing or nonequilibrium pH gradient electrophoresis, followed by PAGE. Serum samples from 15 infertile male subjects and 6 infertile female subjects that contained ASA as assayed by the immunobead binding test (IBT) were analyzed by Western blotting followed by enhanced chemiluminescence (ECL). Serum samples from 10 fertile subjects (5 males and 5 females) that were ASA negative by IBT were used as controls. The ECL blots were analyzed by computer scanning to compare the immunoreactivity between serum samples from fertile and infertile subjects and to identify the antigens unique to the sera of the infertile subjects; 98 sperm auto- and iso-antigenic protein spots were recognized by sera from infertile males and females but not from fertile subjects. Based on vectorial labeling with 125I at the sperm surface, a subset of 6 auto- and iso-antigens was identified as possibly relevant to antibody-mediated infertility.     

Separation and identification of soybean leaf proteins by two-dimensional gel electrophoresis and mass spectrometry

To establish a proteomic reference map for soybean leaves, we separated and identified leaf proteins using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and mass spectrometry (MS). Tryptic digests of 260 spots were subjected to peptide mass fingerprinting (PMF) by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) MS. Fifty-three of these protein spots were identified by searching NCBInr and SwissProt databases using the Mascot search engine. Sixty-seven spots that were not identified by MALDI-TOF-MS analysis were analyzed with liquid chromatography tandem mass spectrometry (LC-MS/MS), and 66 of these spots were identified by searching against the NCBInr, SwissProt and expressed sequence tag (EST) databases. We have identified a total of 71 unique proteins. The majority of the identified leaf proteins are involved in energy metabolism. The results indicate that 2D-PAGE, combined with MALDI-TOF-MS and LC-MS/MS, is a sensitive and powerful technique for separation and identification of soybean leaf proteins. A summary of the identified proteins and their putative functions is discussed.     

Proteome analysis reveals elevated serum levels of clusterin in patients with preeclampsia

Preeclampsia is a pregnancy-specific syndrome and a major cause of maternal mortality. The pathophysiology of preeclampsia is unknown, and no proteome analysis of preeclampsia has been reported. We sought to identify proteins associated with preeclampsia using a proteomic technique and performed two-dimensional electrophoresis (2-DE) on sera from six patients with preeclampsia and six normal pregnant women, followed by comparison of the SYPRO Ruby-stained 2-DE profiles. A group of overexpressed spots was identified in the limited study set. Overexpressed spots were identified as clusterin by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) followed by peptide mass fingerprinting, a protein database search, and Western blot analysis. Additionally, sera of 80 preeclamptic women and 80 normal pregnant women were processed by immunoassay methods to confirm changes in clusterin concentrations quantitatively. Immunoassays showed that clusterin levels in the 80 preeclamptic women were significantly higher than those in the 80 controls (mean +/- SD; 1.62 +/- 0.46 times reference level in preeclamptic women vs. 1.30 +/- 0.46 times reference level in controls, P < 0.001). Proteomic analysis of serum proteins is a promising tool for studying preeclampsia pathophysiology and identifying proteins associated with preeclampsia.     

柔嫩艾美耳球虫地克株利抗药株与敏感株孢子化卵囊的蛋白质差异分析

利用双向电泳技术,对本实验室诱导保存的柔嫩艾美耳球虫地克株利抗药株与敏感株的蛋白质表达图谱进行差异比较和分析,发现两者之间差异有5个蛋白质斑点,利用MALDI_TOF_TOF质谱技术对其中4个差异明显的蛋白质斑点进行分析鉴定,获得4个明确的肽质量指纹图谱,通过在NCBInr数据库中检索分析,确定了其中2个蛋白质分别为球虫子孢子表面抗原TA4和热休克蛋白Hsp70 ,另外两种为真核细胞的功能蛋白。上述蛋白的鉴定将对球虫的抗药性产生机理和柔嫩艾美耳球虫地克株利抗药株的分子标志物提供了研究方向。     

Comparison of protein solubilization methods suitable for proteomic analysis of soybean seed proteins

Extraction of soybean seed proteins for two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and mass spectrometry analysis is challenging and inconsistent. In this study, we compared four different protein extraction/solubilization methods-urea, thiourea/urea, phenol, and a modified trichloroacetic acid (TCA)/acetone-to determine their efficacy in separating soybean seed proteins by 2D-PAGE. In all four methods, seed storage proteins were well separated by 2D-PAGE with minor variations in the intensity of the spots. The thiourea/urea and TCA methods showed higher protein resolution and spot intensity of all proteins compared with the other two methods. In addition, several less abundant and high molecular weight proteins were clearly resolved and strongly detected using the thiourea/urea and TCA methods. Protein spots obtained from the TCA method were subjected to mass spectrometry analysis to test their quality and compatibility. Fifteen protein spots were selected, digested with trypsin, and analyzed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and liquid chromatography mass spectrometry (LC-MS). The proteins identified were beta-conglycinin, glycinin, Kunitz trypsin inhibitor, alcohol dehydrogenase, Gly m Bd 28K allergen, and sucrose binding proteins. These results suggest that the thiourea/urea and TCA methods are efficient and reliable methods for 2D separation of soybean seed proteins and subsequent identification by mass spectrometry.     

Extracellular proteome changes of Deinococcus radiodurans under gamma-irradiation stress conditions

To analysis the change of Deinococcus radiodurans extracellular proteins recovering from gamma-irradiation, we examined extracellular proteome changes using two-dimensional polyacrylamide gel electrophoresis. Twenty-six spots on the gel of irradiated sample were showed significant changes compared with spots on the control gel. Using peptide mass fingerprinting via matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS), 21 different proteins could be distinguished. Among the identified proteins, seven are classified in transport and metabolism, and one is involved in intracellular trafficking and secretion. The other proteins are known to several functions in the cytosol. Most of the proteins have not previously been reported to be relevant to radioresistance. These results imply that the transmembrane transportation is involved in and contributes to the radioresistance in this organism.     

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, includ-ing metabolic enzymes, skeleton proteins, heat shock pro-teins, 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.     

Alteration in Endometrial Proteins during Early- and Mid-Secretory Phases of the Cycle in Women with Unexplained Infertility

Background

Compromised receptivity of the endometrium is a major cause of unexplained infertility, implantation failure and subclinical pregnancy loss. In order to investigate the changes in endometrial protein profile as a cause of unexplained infertility, the current study was undertaken to analyze the differentially expressed proteins of endometrium from early-secretory (LH+2) to mid-secretory phase (LH+7), in women with unexplained infertility.

Methods

2-D gel electrophoresis was performed to analyze the proteomic changes between early- (n = 8) and mid-secretory (n = 8) phase endometrium of women with unexplained infertility. The differentially expressed protein spots were identified by LC-MS analysis and validated by immunoblotting and immuno-histochemical analysis in early- (n = 4) and mid-secretory (n = 4) phase endometrium of infertile women. Validated proteins were also analyzed in early- (n = 4) and mid-secretory (n = 4) phase endometrium of fertile women.

Results

Nine proteins were found to be differentially expressed between early- and mid- secretory phases of endometrium of infertile women. The expression of Ras-related protein Rap-1b, Protein disulfide isomerase A3, Apolipoprotein-A1 (Apo-A1), Cofilin-1 and RAN GTP-binding nuclear protein (Ran) were found to be significantly increased, whereas, Tubulin polymerization promoting protein family member 3, Superoxide dismutase [Cu-Zn], Sorcin, and Proteasome subunit alpha type-5 were significantly decreased in mid- secretory phase endometrium of infertile women as compared to early-secretory phase endometrium of infertile women. Validation of 4 proteins viz. Sorcin, Cofilin-1, Apo-A1 and Ran were performed in separate endometrial biopsy samples from infertile women. The up-regulated expression of Sorcin and down-regulated expression of Cofilin-1 and Apolipoprotein-A1, were observed in mid-secretory phase as compared to early-secretory phase in case of fertile women.

Conclusions

De-regulation of the expression of Sorcin, Cofilin-1, Apo-A1 and Ran, during early- to mid-secretory phase may have physiological significance and it may be one of the causes for altered differentiation and/or maturation of endometrium, in women with unexplained infertility.     

Detection of eight antibodies in cancer patients' sera against proteins derived from the adenocarcinoma A549 cell line using proteomics-based analysis

To screen cancer for specific autoantibodies, we applied the approach established by Brichory et al., who reported annexins I and II as specific antigens. Solubilized proteins from a cancer cell line (A549) were separated using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), followed by Western blotting (WB) analysis, in which the sera of individual patients were tested for primary antibodies. We found 11 positive spots on PVDF membrane using a WB/enhanced chemiluminescence detection Kit, and identified eight proteins, such as alpha-enolase, inosine-5'-monophosphate dehydrogenase, aldehyde dehydrogenase, 3-phosphoglycerate dehydrogenase, 3-oxoacid CoA transferase, chaperonin, peroxiredoxin 6 and triosephosphate isomerase, that reacted with these antibodies in patients' sera using MALDI-TOF/TOF. All eight antibodies were not detected in the sera derived from lung tuberculosis and healthy controls.     

人肺鳞癌组织的血清蛋白质组学的比较分析

采用以肿瘤免疫学与蛋白质组学(proteomics)研究技术有机地结合为基础的血清蛋白质组学研究体系(serologicproteomeanalysis ,SERPA)筛选肺癌分子标志物.对10例人肺鳞癌组织,应用双向凝胶电泳(two dimensionalelectrophoresis ,2 DE)技术对同一肺鳞癌组织的细胞总蛋白同时进行电泳后获得3张相同的凝胶,其中一块2 DE凝胶经银染显色作为平行胶,其余两块2 DE凝胶经电转膜将凝胶中的蛋白质转至硝酸纤维素(NC)膜上,然后分别与肺癌患者的自身血清以及正常对照血清进行Western印迹分析,获取Western印迹反应图谱.经计算机图像分析识别差异反应的蛋白质,然后与平行胶比较找出相应的差异反应蛋白质点.获得了分辨率较高的人肺鳞癌组织与患者的自身血清以及正常对照血清的Western印迹反应图谱;图像分析共识别36±8个差异反应的蛋白质;在平行胶上找到了匹配的差异反应蛋白质点.对2 0个差异蛋白质点进行了肽质指纹图分析,鉴定出14个与细胞生长增殖、细胞代谢、细胞周期调控、信号转导等有关的肺鳞癌相关抗原.通过血清蛋白质组技术对肺鳞癌组织进行的研究,建立了分辨率较高的人肺鳞癌组织与患者的自身血清以及正常血清的Western印迹反应图谱,成功鉴定14个肺鳞癌相关抗原,为进一步筛选用于肺鳞癌诊断、治疗和预后评估     

Growth phase dependent regulation of protein composition in Rhodopirellula baltica

Growth phase dependent changes of protein composition in the marine bacterium Rhodopirellula baltica were quantitatively monitored by applying the two-dimensional difference gel electrophoresis (2D DIGE) technology. The number of regulated proteins (fold changes in protein abundance > absolute value(2)) increased from early (10) to late stationary growth phase (179), with fold changes reaching maximal values of 40. About 110 of these regulated protein spots were analysed by MALDI-TOF-MS and identified by mapping of peptide masses. Results indicate an opposing regulation of tricarboxylic acid cycle and oxidative pentose phosphate cycle, a downregulation of several enzymes involved in amino acid biosynthesis and an upregulation of the alternative sigma factor sigmaH in stationary phase. Interestingly, 26 proteins of unknown function were up- or downregulated in the stationary phase. Several proteins were specifically regulated during growth on solid surface (agar plates). These proteins could possibly be involved in the development of the different R. baltica morphotypes, i.e. motile swarmer cells and sessile cell aggregates (so-called rosettes).     

Anticardiolipin antibodies in women with unexplained infertility

Concept of autoimmune basis of infertility is still controversial, particularly regarding the presence of non-organ specific autoantibodies. Non-organ specific anticardiolipin (aCL) and antithyroglobulin (TgAt) antibodies were detected in infertile women. Both partners were evaluated according to the criteria of The American Society for Reproductive Medicine. All the results were normal in cases of unexplained infertility. Antisperm antibodies (ASA) were determined by a mixed antiglobulin reaction (MAR) and the Kibrick agglutination assay, aCL by commercial ELISA, TgAt by commercial RIA. Fertile women had children. Subjects were grouped in fertile (n=27), infertile (n=65), and cases of unexplained infertility (n=42). In fertile women, aCL was below the negative cut-off value (100 %), while women with unexplained infertility were positive in 23.8 %. Anticardiolipin (aCL) antibodies were detected in 21.5 % of infertile patients, most of them with unexplained infertility (15.4 %). Other positive women had partners with ASA (4.6 %), or exhibited a negative postcoital test (1.5 %). In this study aCL were not detected in women with ASA. TgAt incidence was increased in infertile (20 %) and unexplained infertility group (21.4 %) compared to the fertile controls (18.5 %). Increased incidence of aCL and TgAt in infertile women supports the contention that these autoantibodies contribute to the infertility     

Heterogeneous nuclear ribonucleoproteins C1/C2 identified as autoantigens by biochemical and mass spectrometric methods

The antigenic specificity of an unusual antinuclear antibody pattern in three patient sera was identified after separating HeLa-cell nuclear extracts by two-dimensional (2D) gel electrophoresis and localizing the antigens by immunoblotting with patient serum. Protein spots were excised from the 2D gel and their contents were analyzed by matrix-assisted laser desorption-ionization (MALDI) or nanoelectrospray ionization time-of-flight (TOF) tandem mass spectrometry (MS) after in-gel digestion with trypsin. A database search identified the proteins as the C1 and C2 heterogeneous nuclear ribonucleoproteins. The clinical spectrum of patients with these autoantibodies includes arthritis, psoriasis, myositis, and scleroderma. None of 59 patients with rheumatoid arthritis, 19 with polymyositis, 33 with scleroderma, and 10 with psoriatic arthritis had similar antibodies. High-resolution protein-separation methods and mass-spectrometric peptide mapping in combination with database searches are powerful tools in the identification of novel autoantigen specificities.     

Proteomic and immunoproteomic analysis of Aggregatibacter actinomycetemcomitans JP2 clone strain HK1651

The proteome of Aggregatibacter actinomycetemcomitans HK1651 (JP2 clone) and immunoreactive antigens were studied by two-dimensional (2D) gel electrophoresis, matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF-MS), and 2D immunoblotting. The highly leukotoxic JP2 clone of A. actinomycetemcomitans is strongly associated with aggressive periodontitis (AgP) in adolescents of North-West African descent and the pathogenicity of this bacterium is of major interest. Hence, we developed a comprehensive 2D proteome reference map of A. actinomycetemcomitans proteins with 167 identified spots representing 114 different proteins of which 15 were outer membrane proteins. To unravel immunoreactive antigens, we applied 2D-gel and subsequent immunoblotting analyses using sera from five individuals with A. actinomycetemcomitans infections and one healthy control. The analysis revealed 32 immunoreactive proteins. Antibodies to two outer membrane proteins, YaeT (85 kDa) and Omp39 (39 kDa), not previously described as immunoreactive, were found only in subjects with current or previous A. actinomycetemcomitans JP2 infection. Further proteome-based studies of A. actinomycetemcomitans combined with analyses of the humoral immune response and targeted against outer membrane proteins may provide important insight into the host relationship of this important pathogen.     

Nondenaturing two-dimensional electrophoresis enzyme profile involving activity and sequence structure of cytosol proteins from mouse liver

After cytosol proteins in the mouse liver were separated by nondenaturing two-dimensional electrophoresis (2-DE), activities of several enzymes, such as fructose bisphosphatase, sorbitol dehydrogenase and malate dehydrogenase, transferase and sorbitol dehydrogenase, or several dehydrogenases, were analyzed on the same 2-D gel. Further, peptidase (or protease) activity can be examined by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) when peptides such as angiotensin and adenocorticotropic hormone are incubated in the presence of the cytosol protein separated by nondenaturing 2-DE. Sequence structures of proteins on the 2-D gel were analyzed by peptide mass fingerprinting using MALDI-TOF-MS or by peptide sequencing using electrospray ionization-tandem mass spectrometry (ESI-MS/MS). The combination of activity and sequence structure accurately verified the position and activity range of the separated enzymes on the nondenaturing 2-D gel. From these results, we created a nondenaturing 2-D enzyme profile involving activities and sequence structure of cytosol proteins from mouse liver. This profile can be used for checking whether activities of enzymes were specifically or nonspecifically inhibited by inhibitors.     

Proteomic analysis of maternal serum in down syndrome: identification of novel protein biomarkers

Down syndrome (DS) is the most prevalent chromosomal disorder, accounting for significant morbidity and mortality. Definitive diagnosis requires invasive amniocentesis, and current maternal serum-based testing requires a false-positive rate of about 5% to detect 85% of affected pregnancies. We have performed a comprehensive proteomic analysis to identify potential serum biomarkers to detect DS. First- and second-trimester maternal serum samples of DS and gestational age-matched controls were analyzed using multiple, complementary proteomic approaches, including fluorescence 2-dimensional gel electrophoresis (2D-DIGE), 2-dimensional liquid chromatography-chromatofocusing (2D-CF), multidimensional protein identification technology (MudPIT; LC/LC-MS/MS), and MALDI-TOF-MS peptide profiling. In total, 28 and 26 proteins were differentially present in first- and second-trimester samples, respectively. Of these, 19 were specific for the first trimester and 16 for the second trimester, and 10 were differentially present in both trimesters. Analysis of MALDI-TOF-MS peptide profiles with pattern-recognition software also discriminated between DS and controls in both trimesters, with an average recognition capability approaching 96%. A majority of the biomarkers identified are serum glycoproteins that may play a role in cellular differentiation and growth of fetus. Further characterization and quantification of these markers in a larger cohort of subjects may provide the basis for new tests for improved DS screening.     

Proteomics Approaches for Identification of Tumor Relevant Protein Targets in Pulmonary Squamous Cell Carcinoma by 2D-DIGE-MS

Potential markers for progression of pulmonary squamous cell carcinoma (SCC) were identified by examining samples of lung SCC and adjacent normal tissues using a combination of fluorescence two-dimensional difference gel electrophoresis (2D-DIGE), matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS), and electrospray ionization quadrupole-time of flight mass spectrometry (ESI-Q-TOF). The PANTHER System was used for gel image based quantification and statistical analysis. An analysis of proteomic data revealed that 323 protein spots showed significantly different levels of expression (P≤0.05) in lung SCC tissue compared to expression in normal lung tissue. A further analysis of these protein spots by MALDI-TOF-MS identified 81 different proteins. A systems biology approach was used to map these proteins to major pathways involved in numerous cellular processes, including localization, transport, cellular component organization, apoptosis, and reproduction. Additionally, the expression of several proteins in lung SCC and normal tissues was examined using immunohistochemistry and western blot. The functions of individual proteins are being further investigated and validated, and the results might provide new insights into the mechanism of lung SCC progression, potentially leading to the design of novel diagnostic and therapeutic strategies.     

2D-DiGE analysis of the human endometrial secretome reveals differences between receptive and nonreceptive states in fertile and infertile women

Endometrial secretions in the uterine cavity contain mediators important for endometrial receptivity and embryo implantation. Unbiased analysis of uterine fluid from a receptive versus nonreceptive time of the menstrual cycle and in fertile and infertile women will provide new insights into uterine receptivity. We hypothesized that proteomic analysis of human uterine lavages would identify proteins important for the establishment of pregnancy in humans. Lavages collected from fertile (n = 7) and infertile (n = 8) women during the midsecretory (MS) phase, and from fertile women during the midproliferative (MP) (n = 7) phase, were assessed using 2D-differential in gel electrophoresis (2D-DiGE) over a pI 4-7 range. Statistical analysis revealed 7 spots that were significantly decreased in the MP compared to the MS phase, while 18 spots showed differential expression between fertile and infertile women. A number of proteins were identified by mass spectrometry, including antithrombin III and alpha-2-macroglobulin, whose production was confirmed in endometrial epithelium. Their staining pattern suggests roles during embryo implantation. Assessment of the human endometrial secretome has identified differences in the protein content of uterine fluid with respect to receptivity and fertility.