Establishment of a near-standard two-dimensional human urine proteomic map

A proteomic map for human urine on two-dimensional (2-D) gels has been developed. Initial studies demonstrated that the urine proteins prepared by conventional methods showed interference and poor reproducibility in 2-D electrophoresis (2-DE). To address this issue, urine samples were dialyzed to remove any interfering molecules. The dialysis of urine proteins and the concentration by lyophilization without fractionation significantly improved the reproducibility and resolution and likely represents the total urine proteins on a 2-D gel. In addition, removing albumin from urine using Affi-Gel Blue helped to identify the low-abundant proteins. Using the developed method, we prepared proteins from urine collected from healthy females and males. The large inter- and intra-subject variation in protein profiles on 2-D gels made it difficult to establish a normal human urine proteomic 2-D map. To resolve this problem, urinary proteins were prepared from the pooled urine collected from 20 healthy females and males, respectively. The established male and female urine proteomes separated on 2-D gels were almost identical except for some potential sex-dependent protein spots. We have annotated 113 different proteins on the 2-D gel by peptide mass fingerprinting (PMF). We propose that the established total urine proteome can be used for 2-DE analysis, liquid chromatography-tandem mass spectrometry (LC-MS/MS), and identification of novel disease-specific biomarkers.     

蛋白负染方法在蛋白质组学中的应用评价

为评价蛋白质负染方法在蛋白质组学分析中的应用,采用负染和考马斯亮蓝染色两种方法对同一样品的双向电泳胶进行染色,取相对应的8对蛋白点,并进行胶内酶解及MALDI-TOF/TOF分析,比较两种方法与质谱的兼容性。图像分析显示,负染方法展示出的蛋白点更多,但三维峰图不如考染明晰;质谱结果显示,8个负染蛋白点中有7个鉴定结果有效,8个考染蛋白点鉴定结果均有效。因此可以得出以下结论:负染的灵敏度高于考染,与质谱的兼容性良好,适用于建立双向电泳参考图谱的研究;但负染后的胶图不适于进行蛋白点丰度对比分析。     

Proteomic analysis of Holm oak (Quercus ilex subsp. ballota [Desf.] Samp.) pollen

This paper presents an analysis of Holm oak pollen proteome, together with an evaluation of the potentiality that a proteomic approach may have in the provenance variability assessment. Proteins were extracted from pollen of four Holm oak provenances, and they were analyzed by gel-based (1- and 2-DE in combination with MALDI-TOF/TOF) and gel-free (nLC-LTQ Orbitrap MS) approaches. A comparison of 1- and 2-DE protein profiles of the four provenances revealed significant differences, both qualitative and quantitative, in abundance (18 bands and 16 spots, respectively). Multivariate statistical analysis carried out on bands and spots clearly showed distinct associations between provenances, which highlight their geographical origins. A total of 100 spots selected from the 402 spots observed on 2-DE gels were identified by MALDI-TOF/TOF. Moreover, a complementary gel-free shotgun approach was performed by nLC-LTQ Orbitrap MS. The identified proteins were classified according to biological processes, and most proteins in both approaches were related to metabolism and defense/stress processes. The nLC-LTQ Orbitrap MS analysis allowed us the identification of proteins belonging to the cell wall and division, transport and translation categories. Besides providing the first reference map of Holm oak pollen, our results confirm previous studies based on morphological observations and acorn proteomic analysis. Moreover, our data support the valuable use of proteomic techniques as phylogenetic tool in plant studies.     

Protein/RNA coextraction and small two-dimensional polyacrylamide gel electrophoresis for proteomic/gene expression analysis of renal cancer biopsies

A small amount of bioptic tissue ( approximately 5-10mg of fresh tissue) usually does not contain enough material to extract protein and RNA separately, to obtain preparative two-dimensional polyacrylamide gel electrophoresis (2-DE), and to identify a large number of separated proteins by MS. We tested a method, on small renal cancer specimens, for the coextraction of protein and RNA coupled with 2-DE and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) or quadrupole time-of-flight (Q-TOF) analysis. We coextracted 0.28+/-0.05mg of proteins and 2.5+/-0.33microg of RNA for each 10mg of renal carcinoma tissue. Small and large 2-DE gels were compared: they showed a similar number of spots, and it was possible to match each other; using small format gels, one-fifth of the protein amount was required to identify, by Q-TOF analysis, the same number of proteins identifiable in large-format gel using MALDI-TOF analysis. Quality of RNA coextracted with the proteins was tested by real-time PCR on a set of housekeeping genes. They were quantified with high amplification efficiency and specificity. In conclusion, using 5 to 10mg of fresh tissue, it was possible to perform comprehensive parallel proteomic and genomic analysis by high-resolution, small-format 2-DE gels, allowing approximately 300 proteins identification and 1000 genes expression analysis.     

Comparative Proteome Analysis of Human Temporal Cortex Lobes by Two-Dimensional Electrophoresis and Identification of Selected Common Proteins

Human brain proteins were isolated from left and right temporal cortex lobes at the age of 73, 23, 84 years and separated by two-dimensional gel electrophoresis (2-DE). 2-DE was carried out with an immobilized pH gradient strip in the first dimension and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the second dimension. Over 800 polypeptide spots were resolved with a silver-staining protocol by computerized 2-D gel analsis. Seven of the polypeptide spots were evidently distinguishable between human left and right temporal lobes. Four of the polypeptide spots were larger and three were smaller in human right temporal lobe. One of these three protein spots that have descendent expression in human right temporal lobe was identified as carbonyl reductase (NADPH) 1 by MALDI-TOF MS. Thirty-three common spots were identified by ESI-MS/MALDI-TOF MS/Edman sequencing and a protein database search. These identified proteins include some important enzymes and regulating proteins.     

Protein expression of lymphocytes in HLA-DR transgenic pigs by a proteomic approach

Matching donor and recipient human leucocyte antigen (HLA-II) could conquer cell-mediated rejection following transplantation. Transgenic pigs carrying HLA genes that "humanize" porcine organs, tissues, and cells were successfully generated. This study further clarifies the effect of HLA-DR transgenes on lymphocyte protein expression, via a proteomic approach. Lymphocytes were isolated from two HLA-DR transgenic pigs and three nontransgenic littermates on 157 d after birth. Soluble protein of 1x10(7) cells was separated using 2-DE. In total, 301 colloidal CBB-stained protein spots detected on all five 2-D gels were quantified. Thirty-three proteins were differentially expressed by a factor of 1.5. These proteins were subsequently identified by MALDI-TOF MS and MALDI-TOF/TOF MS/MS. These proteins were sorted into the following categories: chaperones, T-lymphocyte function, DNA/RNA processing, cytoskeleton-associated proteins, signal transduction, enzymes, and unknown. Previous studies have suggested that some of the identified proteins are associated with lymphocyte activation/proliferation. The identities of the unidentified spots and the systematic effect of these up- and down-regulated proteins on T-cell function in HLA-DR transgenic pigs require further exploration.     

Proteomic analysis of human osteoarthritic chondrocytes reveals protein changes in stress and glycolysis

Osteoarthritis (OA) is characterized by cartilage degradation. The chondrocyte is the only cell type present in mature cartilage, and it is important in the control of cartilage integrity. The aim of this study was to analyze, by a proteomic approach, the changes that are characteristic of OA chondrocytes, and to identify new OA-related proteins. Chondrocytes were isolated from the cartilage of ten OA patients undergoing joint replacement and ten donors with no history of joint disease. Whole-cell proteins were resolved by 2-DE and stained with SYPRO Ruby. Protein expression patterns of 2-DE gels from OA and normal chondrocyte proteins were analyzed with PDQuest 7.3.1 software. OA-related proteins were identified by MALDI-TOF or MALDI-TOF/TOF MS. The results were validated for ANXA1, GSTO1, GRP78, and HSP90beta in cells by Western blotting and in tissue cartilage by immunohistochemistry. Results showed an average of 700 protein spots that were present in the 2-DE gels. Compared to normal chondrocytes, 19 protein spots were found to be significantly increased in OA cells (ratio OA:N> or =2.0, p<0.05), whereas nine were decreased in OA chondrocytes (ratio OA:N< or =0.5, p<0.05). Three stress response proteins were increased (HSP90beta, GRP78, and GRP94) and three proteins involved in glycolysis were decreased (enolase, glyceraldehyde 3-phosphate dehydrogenase, and fructose biphosphate aldolase). Functionally, almost all proteins could be classified as proteins involved in cellular metabolism (33%), structure (21%), or protein targeting (21%).     

Proteome analysis of Nelore bull (Bos taurus indicus) seminal plasma

The Nelore bull (Bos taurus indicus) seminal plasma proteome was analyzed by MALDI-TOF MS and two-dimensional gel electrophoresis. A total of 260 spots were visualized in the 2-DE gel (pI range 3-10) and 13 spots could be identified by peptide mass fingerprinting corresponding to 11 different polypeptides. The results allowed the creation of the first proteomic map of Bos taurus indicus seminal plasma. The roles of the identified proteins in the bull seminal plasma are discussed.     

The alterations of mouse plasma proteins during septic development

A fundamental issue for sepsis therapy is to control the development of inflammation at an early stage. With cecal ligation and puncture (CLP) surgery, the mouse model has clearly shown the septic signs triggered by chronic insult. To monitor the plasma proteomic responses to sepsis, the mouse blood was collected at intervals after sham and CLP surgery followed by the sample treatment to remove high abundance serum albumin. The treated mouse plasma proteins were well resolved by two-dimensional electrophoresis (2-DE). The image analysis revealed that these 2-DE spots observed from the sham and the CLP samples 4 h after surgery were comparable, whereas more than 30 different spots appeared on the 2-DE gels between the sham and CLP mouse plasma 24 h after surgery, indicating that some plasma proteins responded to the inflammatory development. These differential spots were verified by MALDI-TOF/TOF MS, resulting in 13 unique sepsis-responsive proteins. More importantly, most of them exhibited multiple spots as difference on the 2-DE gels. Furthermore, these isospots were incubated with PNGase F to eliminate N-linked oligosaccharides on proteins and then evaluated by Western blot as well as mass spectrometry. The results of PNGase F digestion suggested that most sepsis-associated proteins remained in N-glycosylation status but changed their N-glycans during septic development.     

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.     

Comparative Proteomics Analysis of Cerebrospinal Fluid of Patients with Guillain–Barré Syndrome

To better understand the pathophysiologic mechanisms underlying Guillain-Barré syndrome (GBS), Comparative proteomic analysis of cerebrospinal fluid (CSF) between patients with GBS (the experiment group) and control subjects suffering from other neurological disorders (the control group) was carried out using two-dimensional gel electrophoresis (2-DE) technique, in combination with matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) and database searching to determine abnormal CSF proteins in GBS patients. Image analysis of 2-DE gels silver stained revealed that 10 protein spots showed significant differential expression between the two groups of CSF samples. The expression of cystatin C, transthyretin, apolipoprotein E and heat shock protein 70 were decreased. However, haptoglobin, alpha-1-antitrypsin, apolipoprotein A-IV and neurofilaments were elevated. The subsequent ELISA measured the concentration of cystatin C and confirmed the result of the proteomic analysis. These identified proteins may be involved in the pathophysiological process of GBS and call for further studying the role of these proteins in the pathogenesis of the disease.     

2-DE-based proteomic analysis of common bean (Phaseolus vulgaris L.) seeds

Common bean (Phaseolus vulgaris L.) is the most important grain legume for direct human consumption. Proteomic studies in legumes have increased significantly in the last years but few studies have been performed to date in P. vulgaris. We report here a proteomic analysis of bean seeds by two-dimensional electrophoresis (2-DE). Three different protein extraction methods (TCA-acetone, phenol and the commercial clean-up kit) were used taking into account that the extractome can have a determinant impact on the level of quality of downstream protein separation and identification. To demonstrate the quality of the 2-DE analysis, a selection of 50 gel spots was used in protein identification by mass spectrometry (MALDI-TOF MS and MALDI-TOF/TOF). The results showed that a considerable proportion of spots (70%) were identified in spite of incomplete genome/protein databases for bean and other legume species. Most identified proteins corresponded to storage protein, carbohydrate metabolism, defense and stress response, including proteins highly abundant in the seed of P. vulgaris such as the phaseolin, the phytohemagglutinin and the lectin-related α-amylase inhibitor.     

Protein identification from two-dimensional gel electrophoresis analysis of Klebsiella pneumoniae by combined use of mass spectrometry data and raw genome sequences

Separation of proteins by two-dimensional gel electrophoresis (2-DE) coupled with identification of proteins through peptide mass fingerprinting (PMF) by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is the widely used technique for proteomic analysis. This approach relies, however, on the presence of the proteins studied in public-accessible protein databases or the availability of annotated genome sequences of an organism. In this work, we investigated the reliability of using raw genome sequences for identifying proteins by PMF without the need of additional information such as amino acid sequences. The method is demonstrated for proteomic analysis of Klebsiella pneumoniae grown anaerobically on glycerol. For 197 spots excised from 2-DE gels and submitted for mass spectrometric analysis 164 spots were clearly identified as 122 individual proteins. 95% of the 164 spots can be successfully identified merely by using peptide mass fingerprints and a strain-specific protein database (ProtKpn) constructed from the raw genome sequences of K. pneumoniae. Cross-species protein searching in the public databases mainly resulted in the identification of 57% of the 66 high expressed protein spots in comparison to 97% by using the ProtKpn database. 10 dha regulon related proteins that are essential for the initial enzymatic steps of anaerobic glycerol metabolism were successfully identified using the ProtKpn database, whereas none of them could be identified by cross-species searching. In conclusion, the use of strain-specific protein database constructed from raw genome sequences makes it possible to reliably identify most of the proteins from 2-DE analysis simply through peptide mass fingerprinting.     

适用于双向电泳分析的水稻悬浮细胞外分泌蛋白提取方法

目的:建立适用于双向电泳分析的水稻悬浮细胞外分泌蛋白提取方法。方法:采用酚抽提结合甲醇醋酸铵沉淀法、三氯乙酸-丙酮沉淀法和硫酸铵沉淀等3种方法制备水稻悬浮细胞外分泌蛋白,并进行双向电泳分析;利用Western印迹对候选方法提取的外分泌蛋白进行纯度检测。另外,还利用质谱技术对从双向电泳胶上随机挑选的9个蛋白点进行测定,并用SignalP 3.0 Server对测定的蛋白点进行信号肽预测。结果:酚抽提结合甲醇醋酸铵沉淀法提取的外分泌蛋白得率最高,且双向电泳图谱清晰,并能检测到最多的蛋白点;Western印迹表明利用该法所提取的外分泌蛋白未被细胞内蛋白质污染。利用质谱技术鉴定了随机挑选的9个蛋白点,SignalP 3.0 Server分析表明其中6个蛋白含有信号肽。结论:酚抽提结合甲醇醋酸铵沉淀法是一种适用于双向电泳分析的水稻悬浮细胞外分泌蛋白提取方法。     

Proteome analysis of hairy root from Panax ginseng C.A. Meyer using peptide fingerprinting, internal sequencing and expressed sequence tag data

As an initial step to the comprehensive proteomic analysis of Panax ginseng C. A. Meyer, protein mixtures extracted from the cultured hairy root of Panax ginseng were separated by two-dimensional polyacrylamide gel electrophoresis (2-DE). The protein spots were analyzed and identified by peptide finger printing and internal amino acid sequencing by 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 MS), respectively. More than 300 protein spots were detected on silver stained two-dimensional (2-D) gels using pH 3-10, 4-7, and 4.5-5.5 gradients. Major protein spots (159) were analyzed by peptide fingerprinting or de novo sequencing and the functions of 91 of these proteins were identified. Protein identification was achieved using the expressed sequence tag (EST) database from Panax ginseng and the protein database of plants like Arabidopsis thaliana and Oryza sativa. However, peptide mass fingerprinting by MALDI-TOF MS alone was insufficient for protein identification because of the lack of a genome database for Panax ginseng. Only 17 of the 159 protein spots were verified by peptide mass fingerprinting using MALDI-TOF MS whereas 87 out of 102 protein spots, which included 13 of the 17 proteins identified by MALDI-TOF MS, were identified by internal amino acid sequencing using tandem mass spectrometry analysis by ESI Q-TOF MS. When the internal amino acid sequences were used as identification markers, the identification rate exceeded 85.3%, suggesting that a combination of internal sequencing and EST data analysis was an efficient identification method for proteome analysis of plants having incomplete genome data like ginseng. The 2-D patterns of the main root and leaves of Panax ginseng differed from that of the cultured hairy root, suggesting that some proteins are exclusively expressed by different tissues for specific cellular functions. Proteome analysis will undoubtedly be helpful for understanding the physiology of Panax ginseng.     

Proteomic characterization of human normal articular chondrocytes: a novel tool for the study of osteoarthritis and other rheumatic diseases

Articular cartilage is composed of cells and an extracellular matrix. The chondrocyte is the only cell type present in mature cartilage, and it is important in the control of cartilage integrity. There is currently a great lack of knowledge about the chondrocyte proteome. To solve this deficiency, we have obtained the first reference map of the human normal articular chondrocyte. Cells were isolated from cartilages obtained from autopsies without history of joint disease. Cultured cells were used to obtain protein extracts which were resolved by 2-DE and visualized by silver nitrate or CBB staining. Almost 200 spots were excised from the gels and analyzed using MALDI-TOF or MALDI-TOF/TOF MS. The analysis leads to the identification of 136 spots that represent 93 different proteins. A significant proportion of proteins are involved in cell organization (26%), energy (16%), protein fate (14%), metabolism (12%), and cell stress (12%). From all the identified proteins, annexins, vimentin, transgelin, destrin, cathepsin D, heat shock protein 47, and mitochondrial superoxide dismutase were more abundant in chondrocytes than in other types of mesenchymal cells such as Jurkat-T cells. As metabolic program of chondrocytes is altered in osteoarthritis and other rheumatic diseases, this proteomic map is an important tool for future studies on these pathologies.     

Characterization of metaproteomics in crop rhizospheric soil

Soil rhizospheric metaproteomics is a powerful scientific tool to uncover the interactions between plants and microorganisms in the soil ecosystem. The present study established an extraction method suitable for different soils that could increase the extracted protein content. Close to 1000 separate spots with high reproducibility could be identified in the stained 2-DE gels. Among the spots, 189 spots representing 122 proteins on a 2-DE gel of rice soil samples were successfully identified by MALDI-TOF/TOF-MS. These proteins mainly originated from rice and microorganisms. They were involved in protein, energy, nucleotide, and secondary metabolisms, as well as signal transduction and resistance. Three characteristics of the crop rhizospheric metaproteomics seemed apparent: (1) approximately one-third of the protein spots could not be identified by MALDI-TOF/TOF/MS, (2) the conservative proteins from plants formed a feature distribution of crop rhizospheric metaproteome, and (3) there were very complex interactions between plants and microorganisms existing in a crop rhizospheric soil. Further functional analysis on the identified proteins unveiled various metabolic pathways and signal transductions involved in the soil biotic community. This study provides a paradigm for metaproteomic research on soil biology.     

Two-dimensional electrophoresis protein profile of the phytopathogenic fungus Botrytis cinerea

Botrytis cinerea is a phytopathogenic fungi causing disease in a number of important crops. It is considered a very complex species in which different populations seem to be adapted to different hosts. In order to characterize fungal virulence factors, a proteomic research was started. A protocol for protein extraction from mycelium tissue, with protein separation by 2-DE and MS analysis, was optimised as a first approach to defining the B. cinerea proteome. Around 400 spots were detected in 2-DE CBB-stained gels, covering the 5.4-7.7 pH and 14-85 kDa ranges. The averages of analytical and biological coefficients of variance for 64 independent spots were 16.1% and 37.5%, respectively. Twenty-two protein spots were identified by MALDI-TOF or ESI IT MS/MS, with some of them corresponding to forms of malate dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase. Two more spots matched a cyclophilin and a protein with an unknown function.     

Analysis of mouse skin reveals proteins that are altered in a diet-induced diabetic state: a new method for detection of type 2 diabetes

In this study, proteomic analysis was performed on the skin of C57BL/6J mice with type 2 diabetes and compared to nondiabetic controls. To induce obesity and subsequent diabetes, mice were placed on a high-fat diet for 16 wk. After 16 wk, both diabetic and nondiabetic control mice were sacrificed and their skin removed for analysis. Following 2-DE, proteomic profiles from the skin samples were quantified using PDQuest software. Out of more than 1000 distinct protein spots, 28 were shown to be significantly altered with 6 being decreased and 22 increased in the diabetic state compared to controls. The 28 protein spots were removed from the gels and analyzed by MALDI-TOF and MS/MS analyses. Protein identifications revealed that 17 of the 28 proteins were involved in energy metabolism (60.7% of changes observed). Collectively, none of the significantly altered proteins had been shown previously to be altered in diabetic skin. This study not only helps to identify proteins found in skin samples of obese mice with type 2 diabetes, but also shows that skin biopsies coupled with proteomic analysis may be useful as a noninvasive method for the diagnosis of hyperinsulinemia and diabetes.