蛋白质组学技术对不同性别中国美利奴细毛羊(军垦型) 皮肤差异蛋白的筛选

旨在了解性别因素对绵羊毛性状的影响。以周岁雄性和雌性中国美利奴羊（军垦型）为研究对象,利用液相色谱-串联质谱联用技术和数据非依赖性采集策略的定量蛋白质组学技术筛选皮肤组织差异表达蛋白,并对筛选获得的差异蛋白进行基因本体（gene ontology,GO）功能注释、京都基因与基因组百科全书（Kyoto encyclopedia of genes and genomes,KEGG）代谢通路和蛋白互作分析。结果显示,共计筛选获得差异表达蛋白674种,其中,280种蛋白表达上调,394种蛋白表达下调;通过进一步分析发现,与皮肤毛囊发育及羊毛表型相关的差异蛋白有43种,上调差异蛋白30种,下调差异蛋白13种。GO注释结果显示,在分子功能方面,差异蛋白在氧结合、硫酸软骨素结合、亚铁血红素结合、谷胱甘肽过氧化物酶活性和转运活性等37个过程显著富集;在生物过程方面,差异蛋白在细胞氧化解毒作用、肌肉收缩调节、Notch信号通路、钙离子跨膜转运和谷胱甘肽新陈代谢等120个过程显著富集;在细胞组分方面,主要富集在肥大细胞颗粒、细胞核、肌质网状组织和内质网等31个过程。KEGG通路结果表明,这些差异蛋白涉及16条信号通路,其中,MAPK、P53信号通路和羊毛生长发育密切相关。蛋白质网络互作结果显示,COL1A1蛋白与MMP2、SPARC、THBS1等差异表达蛋白联系较为紧密,其可能在羊毛生长发育过程中发挥着关键作用。本研究为揭示不同性别绵羊毛性状的分子机制积累了基础数据。     

Effect of Seasonal Change on Testicular Protein Expression in White Roman Geese

The purpose of this study was to investigate the change in protein expression in the testes of ganders at various breeding stages. A total of nine 3-year-old male White Roman ganders were used. The blood and testis samples were collected at the nonbreeding, sexual reactivation, and breeding stages for sex hormone analysis and proteomic analysis, respectively. The testicular weight and serum testosterone observed for ganders at the breeding stage were higher than those for ganders at nonbreeding and sexual reactivation stages (P?<?0.05). There were 124 protein spots differentially expressed in the testes of ganders at various reproductive stages. A total of 107 protein spots of 74 proteins was identified through mass spectrometry. Most of the differentially expressed proteins were responsible for the molecular functions of protein binding (24%) and catalytic activity (16%). A functional pathway analysis suggested that proteins involved in steroidogenesis, metabolism, and spermatogenesis pathways changed in the White Roman geese at various reproductive stages. In conclusion, ganders at various reproductive stages exhibited different levels of testosterone and protein expression in the testes. The varied levels of the proteins might be essential and unique key factors in seasonal reproduction in ganders.     

Proteomic Analysis of the Secretome of Porcine Alveolar Macrophages Infected with Porcine Reproductive and Respiratory Syndrome Virus

Porcine reproductive and respiratory syndrome virus (PRRSV) causes porcine reproductive and respiratory syndrome (PRRS), which is characterized by reproductive failure and respiratory disorders. The secretome of PRRSV‐infected porcine alveolar macrophages (PAMs), which are the primary target cells of PRRSV, was analyzed by label‐free quantitative proteomics to gain a profile of proteins secreted during PRRSV infection. A total of 95 secreted proteins with differentially expressed levels between PRRSV‐ and mock‐infected PAMs was screened. Among these, the expression levels of 49 and 46 proteins were up‐regulated and down‐regulated, respectively, in PRRSV‐infected cell supernatants, as compared with mock‐infected cell supernatants. Bioinformatic analysis revealed that the differentially expressed proteins were enriched in several signaling pathways related to the immune and inflammatory responses, such as the Toll‐like receptor signaling pathway and NF‐kappa B signaling pathway, and involved in a great diversity of biological processes, such as protein binding and localization, as well as immune effector processes. In addition, PRRSV‐infected cell supernatants induced significant expression of inflammatory cytokines in vascular endothelial cells. These findings suggest that the secreted proteins play potential roles in the host immune and inflammatory responses as well as PRRSV replication, thereby providing new insights into cell‐to‐cell communication during PRRSV infection.     

Comparative proteomic analysis of human placenta derived from assisted reproductive technology

The aim of this study was to use proteomics-based approach to examine differences in protein expression in placenta derived from assisted reproductive technology (ART) and normal pregnancy. Using 2-DE we found that, compared with the control group, 12 spots in standard in vitro fertilization group and 18 spots in intracytoplasmic sperm injection group were identified as significantly differentially expressed proteins. Among them, six spots were differentially expressed in both standard IVF and ICSI groups with the same change tendency. Totally, 20 proteins were successfully identified by MALDI TOF/TOF MS, including proteins involved in the membrane traffic, metabolism, nucleic acid processing, stress response and cytoskeleton. Notably, five proteins detected to be differentially expressed in both ART groups were identified as annexin A3, hnRNP C1/C2, alpha-SNAP, FTL and ATP5A. Some of the proteins were confirmed by Western blot and immunohistochemistry analysis. Our study allowed for the initial identification of these proteins related to various functions in placentation with significantly altered abundance in ART groups. The present results reveal that abnormal protein profiles are involved in ART placenta and these differentially expressed proteins may be valuable for the evaluation of potential association between ART treatment and offspring outcome.     

Proteomic analyses of apoplastic proteins from germinating Arabidopsis thaliana pollen

Pollen grains play important roles in the reproductive processes of flowering plants. The roles of apoplastic proteins in pollen germination and in pollen tube growth are comparatively less well understood. To investigate the functions of apoplastic proteins in pollen germination, the global apoplastic proteins of mature and germinated Arabidopsis thaliana pollen grains were prepared for differential analyses by using 2-dimensional fluorescence difference gel electrophoresis (2-D DIGE) saturation labeling techniques. One hundred and three proteins differentially expressed (p value≤0.01) in pollen germinated for 6h compared with un-germination mature pollen, and 98 spots, which represented 71 proteins, were identified by LC-MS/MS. By bioinformatics analysis, 50 proteins were identified as secreted proteins. These proteins were mainly involved in cell wall modification and remodeling, protein metabolism and signal transduction. Three of the differentially expressed proteins were randomly selected to determine their subcellular localizations by transiently expressing YFP fusion proteins. The results of subcellular localization were identical with the bioinformatics prediction. Based on these data, we proposed a model for apoplastic proteins functioning in pollen germination and pollen tube growth. These results will lead to a better understanding of the mechanisms of pollen germination and pollen tube growth.     

Expression of lorelei-like genes in aposporous and sexual Paspalum notatum plants

Gametophytic apomictic plants form non-reduced embryo sacs that generate clonal embryos by parthenogenesis, in the absence of both meiosis and egg-cell fertilization. Here we report the sequence and expression analysis of a lorelei-like Paspalum notatum gene, n20gap-1, which encodes a GPI-anchored protein previously associated with apomixis in this species. Phylogeny trees showed that n20gap-1 was evolutionary related to the Arabidopsis thaliana lorelei genes At4g26466 and At5g56170. The lorelei At4g26466 disruption was shown to be detrimental to sperm cell release in arabidopsis. RFLP (Restriction Fragment Length Polymorphism) analysis revealed the occurrence of several homologous sequences in the Paspalum notatum genome, exhibiting polymorphisms genetically linked to apomixis. Real-time PCR showed that lorelei-family genes present a minor activity peak at pre-meiosis and a major one at anthesis. The apomictic genotype analyzed showed a significantly increased activity at pre-meiosis, post-meiosis and anthesis with respect to a sexual genotype. In situ hybridization assays revealed expression in integuments, nucellus and the egg-cell apparatus. Several n20gap-1 alleles differing mainly at the 3' UTR sequence were identified. Allele-specific real-time PCR experiments showed that allele 28 was significantly induced in reproductive tissues of the apomictic genotype with respect to the sexual genotype at anthesis. Our results indicate that P. notatum lorelei-like genes are differentially expressed in representative sexual (Q4188) and apomictic (Q4117) genotypes, and might play a role in the final stages of the apomixis developmental cascade. However, the association of n20gap-1 expression with the trait should be confirmed in significant number of sexual and apomictic genotypes.     

Comparative proteomic analysis provides new insights into the fiber elongating process in cotton

A comparative proteomic analysis was performed to explore the mechanism of cell elongation in developing cotton fibers. The temporal changes of global proteomes at five representative development stages (5-25 days post-anthesis [dpa]) were examined using 2-D electrophoresis. Among approximately 1800 stained protein spots reproducibly detected on each gel, 235 spots were differentially expressed with significant dynamics in elongating fibers. Of these, 120 spots showed a more than 2-fold change in at least one stage point, and 21 spots appeared to be specific to developmental stages. Furthermore, 106 differentially expressed proteins were identified from mass spectrometry to match 66 unique protein species. These proteins involve different cellular and metabolic processes with obvious functional tendencies toward energy/carbohydrate metabolism, protein turnover, cytoskeleton dynamics, cellular responses and redox homeostasis, indicating a good correlation between development-dependent proteins and fiber biochemical processes, as well as morphogenesis. Newly identified proteins such as phospholipase D alpha, vf14-3-3 protein, small ras-related protein, and GDP dissociation inhibitor will advance our knowledge of the complicated regulatory network. Identification of these proteins, combined with their changes in abundance, provides a global view of the development-dependent protein changes in cotton fibers, and offers a framework for further functional research of target proteins associated with fiber development.     

Two-dimensional liquid chromatography-tandem mass spectrometry coupled with isobaric tags for relative and absolute quantification (iTRAQ) labeling approach revealed first proteome profiles of pulmonary alveolar macrophages infected with porcine reproductive and respiratory syndrome virus

Porcine reproductive and respiratory syndrome (PRRS) has devastated the pig industry worldwide for almost 25 years, and its virus (PRRSV) preferentially infects and replicates in pulmonary alveolar macrophages (PAMs). To discover cellular protein responses in PRRSV-infected PAMs, two-dimensional liquid chromatography-tandem mass spectrometry coupled with isobaric tags for relative and absolute quantification (iTRAQ) labeling was employed to quantitatively identify the differentially expressed proteins between the PRRSV-infected groups and the controls. A total of 160 cellular proteins in PAMs that were significantly altered post-infection were identified. These differentially expressed proteins are related to the biological processes of virus binding, cell structure, signal transduction, cell adhesion, etc., and their interactions. This is the first report that analyzed the cellular protein profile of PRRSV-infected PAMs using iTRAQ technology, and this data provides important information to help understand the host response to PRRSV and to define the cellular requirements for the underlying mechanism of PRRSV replication and pathogenesis.     

Analysis of proteomic changes in roots of soybean seedlings during recovery after flooding

A proteomic approach was used to identify proteins involved in post-flooding recovery in soybean roots. Two-day-old soybean seedlings were flooded with water for up to 3 days. After the flooding treatment, seedlings were grown until 7 days after sowing and root proteins were then extracted and separated using two-dimensional polyacrylamide gel electrophoresis (2-DE). Comparative analysis of 2-D gels of control and 3 day flooding-experienced soybean root samples revealed 70 differentially expressed protein spots, from which 80 proteins were identified. Many of the differentially expressed proteins are involved in protein destination/storage and metabolic processes. Clustering analysis based on the expression profiles of the 70 differentially expressed protein spots revealed that 3 days of flooding causes significant changes in protein expression, even during post-flooding recovery. Three days of flooding resulted in downregulation of ion transport-related proteins and upregulation of proteins involved in cytoskeletal reorganization, cell expansion, and programmed cell death. Furthermore, 7 proteins involved in cell wall modification and S-adenosylmethionine synthesis were identified in roots from seedlings recovering from 1 day of flooding. These results suggest that alteration of cell structure through changes in cell wall metabolism and cytoskeletal organization may be involved in post-flooding recovery processes in soybean seedlings.     

Comparative proteomic analysis of hearts of adult SCNT Bama miniature pigs (Sus scrofa)

This study aims to determine the effects of SCNT on cardiac development of SCNT pigs through proteomic methods. Heart proteins from three adult SCNTs and two normal reproductive Bama miniature pigs were extracted, separated, and identified via comparative proteomic methods, including two-dimensional gel electrophoresis, mass spectrometry, and Western blot. Eleven differentially expressed spots were identified as differentially expressed proteins, of which five spots were upregulated proteins such as cardiac myosin heavy chain, cathepsin D, and heat shock protein beta-1 (HSP27). By contrast, six spots were downregulated proteins such as alpha skeletal muscle and actin. The results also demonstrated that nuclear transfer might result in abnormal expression of some important proteins in hearts from SCNT pigs, and affect the cardiac development in SCNT pigs' survival.     

An Integrated Approach Based on Multiplexed Protein Array and iTRAQ Labeling for In-Depth Identification of Pathways Associated to IVF Outcome

The emergence of high-throughput protein quantification methodologies has enabled the comprehensive characterization by longitudinal and cross-sectional studies of biological fluids under physiological and pathological conditions. In particular, the simultaneous investigation of cytokines and growth factors signaling pathways and their associated downstream effectors by integrated multiplexed approaches offers a powerful strategy to gain insights into biological networks and processes in living systems. A growing body of research indicates that bioactive molecules of human reproductive fluids, including human follicular fluid (hFF), may affect oocyte quality, fertilization and embryo development, thus potentially influencing the physiopathology of pregnancy-related conditions.In this work, an iTRAQ labeling strategy has been complemented with a multiplexed protein array approach to analyze hFFs with the aim to investigate biological processes and pathways related to in vitro fertilization (IVF) outcome. The iTRAQ labeling strategy lead to the quantification of 89 proteins, 30 of which were differentially expressed in hFFs with successful compared to unsuccessful IVF outcome. The targeted study, based on multiplexed antibody protein arrays, allowed the simultaneous quantification of 27 low abundance proteins, including growth factors, chemokines and cytokines endowed with pro- and anti-inflammatory activity. A significant number of differentially regulated proteins were involved in biological functions related to blood coagulation, acute phase response signaling and complement system. Overall, the present results provide an integrated overview of protein changes in hFFs associated to IVF outcome, thus improving current knowledge in reproductive medicine and fertility research.     

Identification of serum biomarkers for premature ovarian failure

Premature ovarian failure (POF) is defined when a female achieves menopause before the age of 40. Although many conditions are known to be causative for POF, the most common one is idiopathic. This study was undertaken to investigate the pathogenesis of POF using proteomic tools. Two-dimensional electrophoresis (2-DE) analysis was performed to screen for proteins differentially expressed in patients with POF. Using liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS), we identified 11 significant proteins differentially expressed in the serum of POF patients: 5 proteins with expression increased more than two folds, 5 proteins with expression decreased more than two folds, and 1 protein expressed specifically in the serum of patients with POF. The results of the 2-DE analysis were further validated by Western blotting and ELISA analyses, which 5 reproductive system-related proteins (Ceruloplasmin, Complement C3, Fibrinogen α, Fibrinogen β, and SHBG) were selected. The different expression levels for these proteins were confirmed and demonstrated the possibility of using them as biomarkers to screen POF. These pre-clinical data provide plausible translational implications for targeting the pathogenesis of POF for each protein.