A reference map of a human pituitary adenoma proteome

In order to compare the proteomes from different cell types of pituitary adenomas for our long-term goal to clarify the molecular mechanisms that participate in the formation of pituitary adenoma, and to detect any tumor-related marker for an "early-stage" diagnosis, the two-dimensional gel electrophoresis (2-DE) reference map of a pituitary adenoma tissue proteome is described here. A vertical, two-dimensional (2-D) polyacrylamide gel electrophoresis system and PDQuest image analysis software have been used to provide a high level of between-gel reproducibility and to accurately array each protein expressed in a pituitary adenoma tissue. Mass spectrometry (matrix-assisted laser desorption/ionization-time of flight MALDI-TOF and liquid chromatography-electrospray ionization-quadrupole-ion trap LC-ESI-Q-IT) and protein databases were used to characterize each protein in the 2-D gel. The results demonstrate that a good reproducibility of the 2-D gel pattern was attained. The position deviation of matched spots among four 2-D gels was 1.95 +/- 0.45 mm in the isoelectric focusing direction, and 1.70 +/- 0.53 mm in the sodium dodecyl sulfate-polyacrylamide gel electrophoresis direction. A total of ca. 1000 protein spots were separated by 2-DE, and 135 protein spots that represent 111 proteins were characterized with mass spectrometry (96 spots for MALDI-TOF, 39 spots for LC-ESI-Q-IT). The characterized proteins include pituitary hormones, cellular signals, enzymes, cellular-defense proteins, cell-structure proteins, transport proteins, etc. Those proteins were located in the cytoplasmic, cellular membrane, mitochondrial, endoplasmic reticulum, nuclear, ribonucleosome, extracellular fractions, or were secreted in plasma, etc. Those identified proteins contribute to a functional profile of the pituitary adenoma proteome. These data will be used to expand the proteome database of the human pituitary, which can be accessed in the website http://www.utmem.edu /proteomics.     

Two-dimensional electrophoresis database of fluorescence-labeled proteins of colon cancer cells

We constructed a novel database of the proteome of DLD-1 colon cancer cells by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) of fluorescence-labeled proteins followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) analysis. The database consists of 258 functionally categorized proteins corresponding to 314 protein spots. The majority of the proteins are oxidoreductases, cytoskeletal proteins and nucleic acid binding proteins. Phosphatase treatment showed that 28% of the protein spots on the gel are phosphorylated, and mass spectrometric analysis identified 21 of them. Proteins of DLD-1 cells and of laser-microdissected colon cancer tissues showed similar distribution on 2D gels, suggesting the utility of our database for clinical proteomics.     

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.     

大鼠脑皮质表达蛋白质组学研究

文章用蛋白质组学方法初步分析大鼠脑皮质蛋白质的表达。提取大鼠脑皮质蛋白质,双向凝胶电泳分离,考马斯亮蓝染色,胰蛋白酶胶内酶解,用基质辅助激光解吸/电离飞行时间质谱对酶解后的肽段进行分析,根据肽质量指纹图谱,检索专业数据库(Swissprot),对蛋白质进行鉴定。鉴定出84个蛋白,分别属于代谢酶、细胞骨架蛋白、热休克蛋白、抗氧化蛋白、信号传导蛋白、蛋白酶体相关蛋白、神经元特异蛋白及神经胶质蛋白等。文章结果丰富了大鼠脑皮质蛋白质组数据库,为在大鼠模型上研究神经疾病奠定了基础。     

Identification of human whole saliva protein components using proteomics

The determination of salivary biomarkers as a means of monitoring general health and for the early diagnosis of disease is of increasing interest in clinical research. Based on the linkage between salivary proteins and systemic diseases, the aim of this work was the identification of saliva proteins using proteomics. Salivary proteins were separated using two-dimensional (2-D) gel electrophoresis over a pH range between 3-10, digested, and then analyzed by matrix assisted laser desorption/ionization-time of flight (MALDI-TOF)-TOF mass spectrometry (MS) and tandem mass spectrometry (MS/MS). Proteins were identified using automated MS and MS/MS data acquisition. The resulting data were searched against a protein database using an internal Mascot search routine. Ninety spots give identifications with high statistical reliability. Of the identified proteins, 11 were separated and identified in saliva for the first time using proteomics tools. Moreover, three proteins that have not been previously identified in saliva, PLUNC, cystatin A, and cystatin B were identified.     

双向电泳结合质谱分析长双歧杆菌XY01分泌蛋白质图谱

菌体的分泌蛋白质在宿主和菌体的相互作用之间起着重要的作用. 本研究采用双向凝胶电泳的方法建立了长双歧杆菌XY01分泌蛋白质图谱,通过MALDI-TOF/TOF质 谱鉴定和数据库搜索,对鉴定到的分泌蛋白进行了分析. 共检测到21个蛋白质点, 成功鉴定18个蛋白质点,分别代表14个不同的蛋白质,等电点分布在4.5～7.0之间 ,分子质量分布在20 ～65 kD之间;通过COGs分类和功能分析,信号肽和细胞定位及KEGG代谢通路分析. 结果表明,这些蛋白质对菌体细胞壁/膜的形成、生物信号传导和物质代谢等起着重要作用. 研究结果为长双歧杆菌蛋白质组学和基因组学的研究提供了参考.     

Comparative proteome analysis of breast cancer and normal breast

Breast cancer is a leading cause of death for women. The underlying molecular mechanism is still not well understood. In this study, two-dimensional gel electrophoresis combined with mass spectrometry was used to analyze changes in the proteome of infiltrating ductal carcinoma compared to normal breast tissue. Ten sets of two-dimensional gels per experimental condition were analyzed and more than 500 spots each were detected. This revealed 39 spots for which expression in breast cancer cells were reproducibly altered more than twofold compared to normal controls (p<0.01). These spots represented 25 different proteins after identification using the database search after mass spectrometry, comprising cell defense proteins, enzymes involved in glycolytic energy metabolism and homeostasis, protein folding and structural proteins, proteins involved in cytoskeleton and cell motility, and proteins involved in other functions. In addition, 28 nondifferentially expressed proteins with different functions were also mapped and identified, which might help to establish a two-dimensional gel electrophoresis reference map of human breast cancer. Our study shows that proteomics offers a powerful methodology to detect the proteins that show different expression patterns in breast cancer tissue and may provide an accurate molecular classification. The differentially expressed proteins may be used as potential candidate markers for diagnostic purposes or for determination of tumor sensitivity to therapy. The functional implications of the identified proteins are discussed.     

Proteome analysis of mouse primary astrocytes

Astrocytes play a role in energy metabolism, neuronal homeostasis and release of neuronal growth factors and several neurotransmitters. They also relate to a variety of brain diseases and contribute to restore brain dysfunction. Although current research has revealed several roles for astrocytes, knowledge on astrocytic protein expression is limited and a systematic and comprehensive proteome study of astrocytes has not been reported so far. We applied a proteomics technique based on two-dimensional gel electrophoresis coupled with mass spectrometry (MALDI-TOF/TOF) and unambiguously identified 301 spots corresponding to 191 individual proteins in primary mouse astrocytes. The identified proteins were from antioxidant, chaperone, cytoskeleton, nucleic acid binding, signaling, proteasomal, hypothetical and miscellaneous proteins. A reference database is provided and proteins were identified in astrocytes specifically and unambiguously for the first time. A reliable analytical tool independent of antibody availability and specificity along with tentative astrocytic marker proteins is described.     

Proteomics in Drosophila melanogaster: first 2D database of larval hemolymph proteins

A proteomic approach was used for the identification of larval hemolymph proteins of Drosophila melanogaster. We report the initial establishment of a two-dimensional gel electrophoresis reference map for hemolymph proteins of third instar larvae of D. melanogaster. We used immobilized pH gradients of pH 4-7 (linear) and a 12-14% linear gradient polyacrylamide gel. The protein spots were silver-stained and analyzed by nanoLC-Q-Tof MS/MS (on-line nanoscale liquid chromatography quadrupole time of flight tandem mass spectrometry) or by Matrix assisted laser desorption time of flight MS (MALDI-TOF MS). Querying the SWISSPROT database with the mass spectrometric data yielded the identity of the proteins in the spots. The presented proteome map lists those protein spots identified to date. This map will be updated continuously and will serve as a reference database for investigators, studying changes at the protein level in different physiological conditions.     

小鼠晶体蛋白质组的双向电泳和质谱鉴定研究

目的应用双向电泳和质谱技术研究5周龄小鼠晶体蛋白质组。方法提取小鼠晶体总蛋白,进行固相pH梯度（IPG）等电聚焦双向电泳,胶体考马斯亮蓝R-250染色,使用PDQuest7．30图像分析软件分析电泳图像。选择主要蛋白点胶上酶解,应用基质辅助激光解析电离飞行时间／飞行时间（MALDI—TOF／TOF）仪器进行串联质谱（MS／MS）鉴定。结果上样量为882μg和190μg时,分别检测370±41蛋白点（n=3）和57±5个蛋白点（n=3）。高上样量能够较好地分离晶体低丰度蛋白,如念珠状纤维结构蛋白BFSP;低上样量可很好地分离高丰度蛋白-晶体蛋白（包括αA、αB;βA1～βA4;βB1～βB3;γA～γF和γS等）。质谱鉴定得到1种细胞骨架蛋白和16种高丰度晶体蛋白。结论双向电泳和质谱技术有效考察了晶体总蛋白质,为分析白内障形成过程中蛋白质的表达改变提供了新的方法和途径。     

An iterative calibration method with prediction of post-translational modifications for the construction of a two-dimensional electrophoresis database of mouse mammary gland proteins

Protein databases serve as general reference resources providing an orientation on two-dimensional electrophoresis (2-DE) patterns of interest. The intention behind constructing a 2-DE database of the water soluble proteins from wild-type mouse mammary gland tissue was to create a reference before going on to investigate cancer-associated protein variations. This database shall be deemed to be a model system for mouse tissue, which is open for transgenic or knockout experiments. Proteins were separated and characterized in terms of their molecular weight (M(r)) and isoelectric point (pI) by high resolution 2-DE. The proteins were identified using prevalent proteomics methods. One method was peptide mass fingerprinting by matrix-assisted laser desorption/ionization-mass spectrometry. Another method was N-terminal sequencing by Edman degradation. By N-terminal sequencing M(r) and pI values were specified more accurately and so the calibration of the master gel was obtained more systematically and exactly. This permits the prediction of possible post-translational modifications of some proteins. The mouse mammary gland 2-DE protein database created presently contains 66 identified protein spots, which are clickable on the gel pattern. This relational database is accessible on the WWW under the URL: http://www.mpiib-berlin.mpg.de/2D-PAGE.     

A proteomics study of in vitro cyst germination and appressoria formation in Phytophthora infestans

A proteomics study using two-dimensional gel electrophoresis (2-DE) and mass spectrometry was performed on Phytophthora infestans. Proteins from cysts, germinated cysts and appressoria grown in vitro were isolated and separated by 2-DE. Statistical quantitative analysis of the protein spots from five independent experiments of each developmental stage revealed significant up-regulation of ten spots on gels from germinated cysts compared to cysts. Five spots were significantly up-regulated on gels from appressoria compared to germinated cysts and one of these up-regulated spots was not detectable on gels from cysts. In addition, one spot was significantly down-regulated and another spot not detectable on the gels from appressoria. The corresponding proteins to 13 of these spots were identified with high confidence using tandem mass spectrometry and database searches. The functions of the proteins that were up-regulated in germinated cysts and appressoria can be grouped into the following categories: protein synthesis (e.g. a DEAD box RNA helicase), amino acid metabolism, energy metabolism and reactive oxygen species scavenging. The spot not detected in appressoria was identified as the P. infestans crinkling- and necrosis-inducing protein CRN2. The identified proteins are most likely involved in the establishment of the infection of the host plant.     

Comparative proteomics of Cannabis sativa plant tissues.

Comparative proteomics of leaves, flowers, and glands of Cannabis sativa have been used to identify specific tissue-expressed proteins. These tissues have significantly different levels of cannabinoids. Cannabinoids accumulate primarily in the glands but can also be found in flowers and leaves. Proteins extracted from glands, flowers, and leaves were separated using two-dimensional gel electrophoresis. Over 800 protein spots were reproducibly resolved in the two-dimensional gels from leaves and flowers. The patterns of the gels were different and little correlation among the proteins could be observed. Some proteins that were only expressed in flowers were chosen for identification by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and peptide mass fingerprint database searching. Flower and gland proteomes were also compared, with the finding that less then half of the proteins expressed in flowers were also expressed in glands. Some selected gland protein spots were identified: F1D9.26-unknown prot. (Arabidopsis thaliana), phospholipase D beta 1 isoform 1a (Gossypium hirsutum), and PG1 (Hordeum vulgare). Western blotting was employed to identify a polyketide synthase, an enzyme believed to be involved in cannabinoid biosynthesis, resulting in detection of a single protein.     

Characterisation of global protein expression by two-dimensional electrophoresis and mass spectrometry: proteomics of Toxoplasma gondii.

The development of tools for the analysis of global gene expression is vital for the optimal exploitation of the data on parasite genomes that are now being generated in abundance. Recent advances in two-dimensional electrophoresis (2-DE), mass spectrometry and bioinformatics have greatly enhanced the possibilities for mapping and characterisation of protein populations. We have employed these developments in a proteomics approach for the analysis of proteins expressed in the tachyzoite stage of Toxoplasma gondii. Over 1000 polypeptides were reproducibly separated by high-resolution 2-DE using the pH ranges 4-7 and 6-11. Further separations using narrow range gels suggest that at least 3000-4000 polypeptides should be resolvable by 2-DE using multiple single pH unit gels. Mass spectrometry was used to characterise a variety of protein spots on the 2-DE gels. Peptide mass fingerprints, acquired by matrix-assisted laser desorption/ionisation-(MALDI) mass spectrometry, enabled unambiguous protein identifications to be made where full gene sequence information was available. However, interpretation of peptide mass fingerprint data using the T. gondii expressed sequence tag (EST) database was less reliable. Peptide fragmentation data, acquired by post-source decay mass spectrometry, proved a more successful strategy for the putative identification of proteins using the T. gondii EST database and protein databases from other organisms. In some instances, several protein spots appeared to be encoded by the same gene, indicating that post-translational modification and/or alternative splicing events may be a common feature of functional gene expression in T. gondii. The data demonstrate that proteomic analyses are now viable for T. gondii and other protozoa for which there are good EST databases, even in the absence of complete genome sequence. Moreover, proteomics is of great value in interpreting and annotating EST databases.     

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. __________ Translated from Acta Biophysica Sinica, 2007, 23 (1): 151–156 [译自: 生物物理学报]     

Comparison of SYPRO Ruby and Deep Purple using commonly available UV transilluminator: wide-scale application in proteomic research

Fluorescent stains are becoming increasingly useful in proteomics research involving protein expression as well as post-translational modification studies and are particularly useful for samples which are expensive and scarce. The fluorescent dyes Deep Purple and SYPRO Ruby are widely used in protein expression studies. Using UV transillumination and Charged Coupled Device (CCD) based imaging system, their relative sensitivity to detect proteins separated by two-dimensional polyacrylamide gel electrophoresis and downstream protein identification by liquid chromatography-tandem mass spectrometry (LC-MS/MS) was compared. Using mouse liver homogenate, we detected a greater number of spots using SYPRO Ruby over Deep Purple stain. However, the number of matched peptides and the percentage of amino acid residues identified for 21 different proteins were comparable suggesting their equivalency for LC-MS/MS identification. In spite of comparable MS compatibility, we recommend the use of SYPRO Ruby for expression proteomics due to its higher sensitivity in detecting protein spots.     

Highlights on the capacities of "Gel-based" proteomics

Gel-based proteomic is the most popular and versatile method of global protein separation and quantification. This is a mature approach to screen the protein expression at the large scale, and a cheaper approach as compared with gel-free proteomics. Based on two independent biochemical characteristics of proteins, two-dimensional electrophoresis combines isoelectric focusing, which separates proteins according to their isoelectric point, and SDS-PAGE, which separates them further according to their molecular mass. The next typical steps of the flow of gel-based proteomics are spots visualization and evaluation, expression analysis and finally protein identification by mass spectrometry. For the study of differentially expressed proteins, two-dimensional electrophoresis allows simultaneously to detect, quantify and compare up to thousand protein spots isoforms, including post-translational modifications, in the same gel and in a wide range of biological systems. In this review article, the limits, benefits, and perspectives of gel-based proteomic approaches are discussed using concrete examples.     

Increased lungkine and chitinase levels in allergic airway inflammation: a proteomics approach

Asthma is a chronic inflammatory disease characterized by pulmonary eosinophilia and airway hyperresponsiveness. Mechanisms underlying the pathogenesis of asthma are still not fully understood. The present study investigated alterations in global protein expression in bronchoalveolar lavage fluid in allergic airway inflammation using a proteomics approach. BALB/c mice sensitized and challenged with ovalbumin developed airway eosinophilia, mucus hypersecretion, elevation of immunoglobulin E, and airway hyperresponsiveness. Lavage fluid proteins from normal and asthmatic mice were resolved by two-dimensional gel electrophoresis, and identified by peptide mass fingerprinting matrix-assisted laser desorption/ionization-time of flight mass spectrometry. A total of 28 protein spots were significantly altered. Several of these proteins were undetectable or at very low levels in normal mice but were significantly increased in airway inflammation. These include lungkine, a recently described chemokine, a family of chitinases including Ym1, Ym2, and acidic mammalian chitinase, gob-5, a protein that mediates mucus secretion, and surfactant protein-D, a C-type lectin capable of modulating inflammatory responses. Overall, proteomics is a powerful tool in unraveling protein expression changes in allergic airway inflammation. The proteins identified in this study may be associated with the pathogenesis of allergic airway inflammation and may also be found useful as surrogate biomarkers for asthma.