Initial analysis of the phosphoproteome of Chinese hamster ovary cells using electrophoresis.

Protein phosphorylation is a common post-translational modification of enormous biological importance. Analysis of phosphorylation at the global level should shed light on the use of this modification to regulate metabolism, signal transduction, and other processes. We have begun a proteomic analysis of phosphorylation using two-dimensional gel electrophoresis. Chinese hamster ovary (CHO) cells were metabolically labeled using 32P-orthophosphate. The proteins were extracted and run on two-dimensional electrophoresis. Gels were stained using colloidal Coomassie stain, dried, and phosphorimaged. The Coomassie stain allowed the observation of 468 individual protein spots. The phosphorimage showed 181 spots. The phosphoproteome of CHO cells therefore comprises around one third as many proteins as the CHO cell abundance proteome. However, the most intense spots in the phosphoproteome usually do not correlate with intense spots in the abundance proteome. We investigated the effects of labeling time, finding that the number of observable spots increases but the relative intensities also change. We also investigated the effects of adding a phosphatase inhibitor during labeling. Finally, we evaluated a phosphoprotein-specific stain (Pro-Q Diamond) in comparison with radiolabeling methods. There is not perfect correlation between radiolabeled phosphoproteins and Pro-Q Diamond-stained phosphoproteins.     

Analysis of the Arabidopsis cell suspension phosphoproteome in response to short-term low temperature and abscisic acid treatment

To shed light on the early protein phosphorylation events involved in plant cell signaling in response to environmental stresses, we studied changes in the phosphorylation status of the Arabidopsis cell suspension proteome after short-term low temperature and abscisic acid (ABA) treatment. We used radioactive pulse-labeling of Arabidopsis cell suspension cultures and two-dimensional (2-D) gel electrophoresis to identify proteins that are differentially phosphorylated in response to these treatments. Changes in the phosphorylation levels of several proteins were detected in response to short-term (5 min or less) cold (4°C) and chilling (12°C) stress and ABA treatment, and we observed that some of these changes were common between these treatments. In addition, we used Pro-Q Diamond phosphoprotein gel stain to study the steady-state protein phosphorylation status under the same treatments. We demonstrated that Pro-Q Diamond effectively stained phosphorylated proteins, however, the overall Pro-Q Diamond 2-D gel staining pattern of proteins extracted from low-temperature and ABA-treated cells was not consistent with the gel patterns obtained by in vivo radioactive labeling of phosphoproteins. These in vivo pulsed-labeling experiments demonstrate that the Arabidopsis phosphoproteome is dynamic in response to short-term low temperature and ABA treatment, and thus represents a strategy for the identification of signaling proteins that could be utilized in the production of chilling or freeze tolerant crop varieties.     

A comparative proteome and phosphoproteome analysis of differentially regulated proteins during fertilization in the self-incompatible species Solanum chacoense Bitt

We have used 2-DE for a time-course study of the changes in protein and phosphoprotein expression that occur immediately after fertilization in Solanum chacoense. The phosphorylation status of the detected proteins was determined with three methods: in vivo labeling, immunodetection, and phosphoprotein-specific staining. Using a pI range of 4-7, 262 phosphorylated proteins could be mapped to the 619 proteins detected by Sypro Ruby staining, representing 42% of the total proteins. Among these phosphoproteins, antibodies detected 184 proteins from which 78 were also detected with either of the other two methods (42%). Pro-Q Diamond phosphoprotein stain detected 111 proteins, of which 76 were also detected with either of the other two methods (68%). The 32P in vivo labeling method detected 90 spots from which 78 were also detected with either of other two methods (87%). On comparing before and after fertilization profiles, 38 proteins and phosphoproteins presented a reproducible change in their accumulation profiles. Among these, 24 spots were selected and analyzed by LC-MS/MS using a hybrid quadrupole-TOF (Q-TOF) instrument. Peptide data were searched against publicly available protein and EST databases, and the putative roles of the identified proteins in early fertilization events are discussed.     

长双歧杆菌NCC2705葡萄糖与乳糖代谢的比较蛋白质组学

[目的]以本实验室前期构建的长双歧杆菌NCC2705菌株蛋白质参考图谱为基础,研究长双歧杆菌发酵乳糖和葡萄糖的比较蛋白质组学.[方法]采用ImageMaster 2D Elite Platnum Version 5.0比较分析3倍以上蛋白差异点;利用MALDI-TOF进行差异蛋白鉴定,每个蛋白质点的肽指纹图谱在长双歧杆菌NCC2705菌株的蛋白质数据库用Mascot进行检索;采用Pro-Q磷酸化试剂进行磷酸化蛋白的染色.[结果]鉴定到31个蛋白表达发生显著变化,在乳糖发酵中14个蛋白上调17个蛋白下调.这些蛋白为亲水性酸性蛋白,它们基因的CAI值均在0.5以上,主要包括糖代谢相关蛋白、应激蛋白、转录和翻译相关蛋白,还有一些未知功能的蛋白.此外,有两个蛋白:转醛缩酶(BL0715,transaldolase,tal)L3蛋白点和丙酮酸激酶(BL0988,pyruvate kinase,pyk)G9蛋白点发生了磷酸化作用.[结论]长双歧杆菌NCC2705在乳糖中生长快于葡萄糖,它们的降解途径是相同的;转醛缩酶和丙酮酸激酶发生了翻译后修饰作用,推测转醛缩酶在43T和47S发生了磷酸化,而丙酮酸激酶在65S发生了磷酸化.     

食源性肥胖大鼠下丘脑差异表达蛋白的蛋白质组学分析

建立食源性肥胖大鼠模型,对正常大鼠和肥胖大鼠下丘脑全蛋白进行双向凝胶电泳,产生下丘脑蛋白双向凝胶电泳图谱.对图谱进行比对分析后,从凝胶上切取差异表达的蛋白点,经胶内酶解,通过基质辅助激光解吸/电离飞行时间质谱(MALDI-TOF-MS) 对酶解后的肽段进行分析,再经数据库（NCBInr）检索,对蛋白质进行鉴定.研究发现,正常组表达图谱可检测到1　160±15（n=5）个蛋白点,肥胖组表达图谱可检测到1　070±10 （n=5）个蛋白点,与对照组相比,匹配率大于80％.并且成功鉴定了17种差异表达蛋白质,其中有7 种在肥胖组表达上调,10种表达下调.它们分别属于代谢酶、细胞周期调控因子、抗氧化蛋白、信号传导蛋白、蛋白酶体相关蛋白、细胞骨架蛋白以及未知蛋白等. 与正常对照组相比,肥胖组的蛋白质表达存在着较大差异,通过对差异表达蛋白的分析,提示了在肥胖发生的过程中,下丘脑神经中枢经历了一个非常复杂的信号活动和特定改变,为深入认识肥胖的发病机制奠定了基础.     

家蚕丝腺蛋白质组学研究方法的建立

通过高精度的双向电泳技术对家蚕中部丝腺组织的蛋白质进行分离,采用基质辅助激光解析电离飞行时间质谱(matrix-assistedlaserdesorption/ionizationtimeofflightmassspectrometry,MALDI-TOF-MS)对其中一些表达量较高的蛋白点进行鉴定,并利用GPMAW(GeneralProtein/MassAnalysisforWindows)软件结合家蚕基因组预测的蛋白质数据库构建本地的肽质量指纹图谱数据库,对所得到的肽质量指纹图谱进行分析。研究发现,经过双向凝胶电泳及其图象分析技术,硝酸银染色和考马斯亮蓝染色分别能分离出500个以上和100个以上的蛋白点。这些蛋白质点主要集中在分子量15~90kD区域,等电点pH3·5~7之间。MALDI-TOF-MS鉴定的25个考染蛋白点中有60%以上的PMF(PeptideMassFingerprint)的信号峰较强。在数据库检索过程中,利用家蚕肽质量指纹数据库所得检索结果与在Mascot的检索结果相比,前者不仅能够准确鉴定出一些已有研究报道的蛋白,从而验证检索方法的可行性,而且还能够对一些已经被家蚕基因组数据库所预测但未曾报道的新蛋白质进行鉴定,从而建立了一整套适合于家蚕蛋白质组研究的方法,并为其它绢丝昆虫蛋白质组研究提供了重要参考。     

A comparative proteomic analysis reveals important proteins for the fertilization and early embryonic development of the oyster Crassostrea gigas

Molluscan development involves important features that are important to understanding not only molluscan ontogeny but also animal evolution. To gain insight into the gamete proteome and protein function in fertilization and early development, we analyzed the proteomes of unfertilized oocytes and early embryos (2/4‐cell stage) of the Pacific oyster, Crassostrea gigas. An oocyte reference map containing 116 protein spots, of which 69 were identified, revealed a high abundance of vitellogenin‐derived protein spots. The differentially regulated protein spots during fertilization were screened using comparative proteomic approaches. In total, 18 differentially regulated protein spots were screened, and 15 of these were identified and divided into three groups. The proteins belonging to the first group function in energy supply and antioxidation and are proposed to ensure successful fertilization by regulating the levels of adenosine triphosphate, resisting oxidative stress, and preventing polyspermy. The proteins of the second group are associated with protein synthesis and modification, reflecting active protein synthesis after fertilization. The three proteins belonging to the final group are hypothesized to function in the regulation of embryonic development through the establishment of cell polarity and modulation of methylation reactions in nuclei. These results will enhance our knowledge of molluscan fertilization and development.     

Analysis of the dynamic Bacillus subtilis Ser/Thr/Tyr phosphoproteome implicated in a wide variety of cellular processes

The physiological role of proteins phosphorylated on serine/threonine/tyrosine (Ser/Thr/Tyr) residues or the identity of the corresponding kinases and phosphatases is generally poorly understood in bacteria. As a first step in analysing the importance of such phosphorylation, we sought to establish the nature of the Ser/Thr/Tyr phosphoproteome in Bacillus subtilis, using in vivo labelling with [(32)P]-orthophosphate, one-unit pH 2-DE, combined with MS. Highly reproducible 2-D profiles of phosphoproteins were obtained with early stationary-phase cells. The 2-D profiles contained at least 80 clearly labelled spots in the pH range 4-7. Forty-six spots were analysed by MS (confirmed in most cases by LC-MS/MS), identifying a total of 29 different proteins, with 19 identified for the first time as bacterial phosphoproteins. These phosphoproteins are implicated in a wide variety of cellular processes, including carbon and energy metabolism, transport, stress and development. Significant changes to the profiles were obtained as a result of cold, heat or osmotic shock, demonstrating that, in stationary-phase cells, the phosphoproteome is dynamic. An initial comparative study indicated that at least 25 [(32)P]-labelled spots were also stained by Pro-Q Diamond, with apparently six additional phosphoproteins uniquely detected by Pro-Q.     

Proteomics reveals elevated levels of PR 10 proteins in saline-tolerant peanut (Arachis hypogaea) calli.

The proteome of a salinity-tolerant Arachis hypogaea L. callus cell line was compared with its sensitive counterpart. Several low molecular weight proteins were detected by two-dimensional electrophoresis as being unique or significantly elevated in the tolerant line. The identities of several of these proteins were established as PR 10 proteins using tandem Mass Spectrometry and are shown to be phosphorylated on the basis of staining with the phosphorylation-specific stain, Pro-Q Diamond. Our results suggest that these differentially phosphorylated PR 10 proteins may play an important role in mediating salinity stress responses.     

Phosphoproteomic analysis of neuronal cell death by glutamate-induced oxidative stress

Oxidative stress is one of the major causes of neuronal cell death in disorders such as perinatal hypoxia and ischemia. Protein phosphorylation is the most significant PTM of proteins and plays an important role in stress-induced signal transduction. Thus, the analysis of alternative protein phosphorylation states which occur during oxidative stress-induced cell death could provide valuable information regarding cell death. In this study, a reference phosphoproteome map of the mouse hippocampal cell line HT22 was constructed based on 125 spots that were identified by MALDI-TOF or LC-ESI-Q-TOF-MS analysis. In addition, proteins of HT22 cells at various stages of oxidative stress-induced cell death were separated by 2-DE and alterations in phosphoproteins were detected by Pro-Q Diamond staining. A total of 17 spots showing significant quantitative changes and seven newly appearing spots were identified after glutamate treatment. Splicing factor 2, peroxiredoxin 2, S100 calcium binding protein A11, and purine nucleoside phosphorylase were identified as up- or down-regulated proteins. CDC25A, caspase-8, and cyp51 protein appeared during oxidative stress-induced cell death. The data in this study from phosphoproteomic analysis provide a valuable resource for the understanding of HT22 cell death mechanisms mediated by oxidative stress.     

Proteome of the bacterium Mycoplasma penetrans

A proteome map of Mycoplasma penetrans has been constructed using two-dimensional gel electrophoresis in combination with mass spectrometry (MS). Mycoplasma penetrans infects the urogenital and respiratory tracts of humans. A total of 207 spots were characterized with MS and, in comparing the experimental data with the DNA sequence-derived predictions, it was possible to assign these 207 spots to 153 genes. The Pro-Q Diamond phosphoprotein dye technology was used for the fluorescent detection of 26 phosphoproteins in the 4-7 pH range.     

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.     

Protein reference mapping of dihydrofolate reductase‐deficient CHO DG44 cell lines using 2‐dimensional electrophoresis

Chinese hamster ovary (CHO) cells are the major mammalian host for producing various therapeutic proteins. Among CHO cells, the dihydrofolate reductase‐deficient CHO DG44 cell line has been used as a popular mammalian host because of the availability of a well‐characterized genetic selection and amplification system. However, this cell line has not been studied at the proteome level. Here, the first detailed proteome analysis of the CHO DG44 cell line is described. A protein reference map of the CHO DG44 cell line was established by analyzing whole cellular proteins using 2‐DE with various immobilized pH gradients (pHs 3–10, 5–8, and 3–6) in the first dimension and a 12% acrylamide gel in the second dimension. The map is composed of over 1400 silver‐stained protein spots. Among them, 179 protein spots, which represent proteins associated with various biological processes and cellular compartments, were identified based on MALDI‐TOF‐MS and MS/MS. This proteome database should be valuable for better understanding of CHO cell physiology and protein expression patterns which may lead to efficient therapeutic protein production.     

AspC基因导入前后E.coli BL21蛋白质组的解析

为了考察表达天冬氨酸转氨酶工程菌在转基因前后蛋白质水平的差异变化,采用固相pH梯度-SDS聚丙烯酰胺双向凝胶电泳对转基因前后的大肠杆菌（E．coli BL21）的总蛋白进行分离,银染、显色后,使用2D蛋白质图象分析系统Image Master 2D Platinum 5．0和SWISS-2D PAGE蛋白质组数据库对双向电泳图谱进行分析,识别了近600个蛋白点,比较分析了与苯丙氨酸合成途径相关的关键蛋白的差异,初步探讨了AspC基因的导入后大肠杆菌蛋白质水平的精细调控。     

Proteome and phosphoproteome dynamic change during cell dedifferentiation in Arabidopsis

Cell dedifferentiation is a cell fate switching process in which a differentiated cell reverts to a status with competence for cell division and organ regeneration like an embryonic stem cell. Although the phenomenon of cell dedifferentiation has been known for over two and a half centuries in plants, little is known of the underlying mechanisms. Here, we have established the proteome map of Arabidopsis cotyledons and investigated the dynamic change of the cotyledon proteome in the time course of cell dedifferentiation. Among the 353 distinct genes, corresponding to 500 2-DE gel protein spots identified with high confidence, 12% have over twofold differential regulations within the first 48 h of induction of cell dedifferentiation. The distributions of these genes among different Gene Ontology categories and gene differential regulations within each of the categories have been examined. In addition, we have investigated the cotyledon phosphoproteome using Pro-Q Diamond Phosphoprotein in Gel Stain followed by mass spectrometry analyses. Among the 53 identified putative phosphoproteins, nine are differentially regulated during cell dedifferentiation. These studies have provided significant new insight into protein and phosphoprotein differential expression during cell dedifferentiation in plants.     

Global quantitative phosphoprotein analysis using Multiplexed Proteomics technology

Systematic parallel analysis of the phosphorylation status of networks of interacting proteins involved in the regulatory circuitry of cells and tissues is certain to drive research in the post-genomics era for many years to come. Reversible protein phosphorylation plays a critical regulatory role in a multitude of cellular processes, including alterations in signal transduction pathways related to oncogene and tumor suppressor gene products in cancer. While fluorescence detection methods are likely to offer the best solution to global protein quantitation in proteomics, to date, there has been no satisfactory method for the specific and reversible fluorescent detection of gel-separated phosphoproteins from complex samples. The newly developed Pro-Q Diamond phosphoprotein dye technology is suitable for the fluorescent detection of phosphoserine-, phosphothreonine-, and phosphotyrosine-containing proteins directly in sodium dodecyl sulfate (SDS)-polyacrylamide gels and two-dimensional (2-D) gels. Additionally, the technology is appropriate for the determination of protein kinase and phosphatase substrate preference. Other macromolecules, such as DNA, RNA, and sulfated glycans, fail to be detected with Pro-Q Diamond dye. The staining procedure is rapid, simple to perform, readily reversible and fully compatible with modern microchemical analysis procedures, such as matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry. Pro-Q Diamond dye technology can detect as little as 1-2 ng of beta-casein, a pentaphosphorylated protein, and 8 ng of pepsin, a monophosphorylated protein. Fluorescence signal intensity correlates with the number of phosphorylated residues on the protein. Through combination of Pro-Q Diamond phosphoprotein stain with SYPRO(R) Ruby protein gel stain, Multiplexed Proteomics technology permits quantitative, dichromatic fluorescence detection of proteins in 2-D gels. This evolving discovery platform allows the parallel determination of protein expression level changes and altered post-translational modification patterns within a single 2-D gel experiment. The linear responses of the fluorescence dyes utilized, allow rigorous quantitation of changes over an unprecedented 500-1000-fold concentration range.