Alteration of protein composition in mouse thymocytes by signals through T-cell receptor

To avoid destructive autoimmunity, T-cell precursors (thymocytes) expressing autoreactive T-cell receptor are deleted in the thymus via an apoptotic process by the signals from the T-cell receptor-CD3 complexes. In order to analyze the apoptotic mechanism, we established a cell-free system using the lysates from mouse thymocytes treated in vivo with anti-CD3 monoclonal antibody (mAb). The soluble cytosolic high molecular mass protein fraction from the anti-CD3-treated thymocytes revealed an activity that directly induces nuclear apoptotic morphological changes and DNA fragmentation. This fragmentation activity was not observed in the fraction from the thymocytes without anti-CD3 treatment. Proteins in both fractions were separated by two-dimensional electrophoresis. The silver-stained gels revealed differences in protein spots. These protein spots were identified by database searching of mass spectrometric (MS) and tandem mass spectrometric (MS/MS) data obtained from in-gel tryptic digests of the spots, using an integrated system of liquid chromatography/electrospray ionization/ion-trap mass spectrometry. In this study, the high mobility group protein HMG2 was identified as one of the cytosolic proteins that is increased by the signals from the T-cell receptor, and heterogeneous nuclear ribonucleoprotein A2/B1 and glyceraldehyde 3-phosphate dehydrogenase were found to be decreased by the signals.     

Two-dimensional electrophoresis analysis of human serum proteins during the acute-phase response.

The serum of patients with meningitis, due to infection by Haemophilus influenzae type b, was analyzed. Several known acute-phase proteins were separated by two-dimensional electrophoresis and estimated quantitatively. In addition, hitherto undescribed reactants were recognized. Gels were calibrated and relevant spots related to master spot numbers in the human serum protein database.     

Identification of platelet proteins separated by two-dimensional gel electrophoresis and analyzed by matrix assisted laser desorption/ionization-time of flight-mass spectrometry and detection of tyrosine-phosphorylated proteins

Different search programs were compared to judge their particular efficiency in protein identification. We established a human blood platelet protein map and identified tyrosine-phosphorylated proteins. The cytosolic fraction of human blood platelets was separated by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and phosphorylated proteins were detected by Western blotting using anti-phosphotyrosine antibodies. Visualized protein spots were excised, digested with trypsin and analyzed by matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). The obtained mass fingerprint data sets have been analyzed using ProFound, MS-Fit and Mascot. For those protein spots with no significant search results MALDI post source decay (PSD) spectra have been acquired on the same sample. For automatic interpretation of these fragment ion spectra, the SEQUEST and Mascot algorithm were applied. Another approach for the identification of phosphorylated proteins is immunoprecipitation using an anti-phosphotyrosine antibody. A method for immunoprecipitation of tyrosine-phosphorylated peptides was optimized.     

Redesign of a dioxygenase in morphine biosynthesis

Opium poppy (Papaver somniferum) produces medicinally important benzylisoquinoline alkaloids, including the analgesics codeine and morphine, in the morphinan pathway. We aligned three dioxygenases that were recently discovered in P. somniferum and subsequently identified the nonconserved regions. Two of these enzymes, codeine O-demethylase (PsCODM) and thebaine O-demethylase (PsT6ODM), are known to facilitate regioselective O-demethylation in morphinan biosynthesis. We systematically swapped the residues that were nonconserved between the PsCODM and PsT6ODM sequences to generate 16 mutant PsCODM proteins that could be overexpressed in Escherichia coli. While wild-type PsCODM can demethylate both codeine and thebaine, one engineered PsCODM mutant selectively demethylates codeine. Use of this reengineered enzyme in the reconstitution of morphine biosynthesis could selectively disable a redundant pathway branch and therefore impact the yields of the downstream products codeine and morphine in subsequent metabolic engineering efforts.     

Proteome analysis of human stomach tissue: separation of soluble proteins by two-dimensional polyacrylamide gel electrophoresis and identification by mass spectrometry

Two-dimensional gel electrophoresis (2-DE) maps for human stomach tissue proteins have been prepared by displaying the protein components of the tissue by 2-DE and identifying them using mass spectrometry. This will enable us to present an overview of the proteins expressed in human stomach tissues and lays the basis for subsequent comparative proteome analysis studies with gastric diseases such as gastric cancer. In this study, 2-DE maps of soluble fraction proteins were prepared on two gel images with partially overlapping pH ranges of 4-7 and 6-9. On the gels covering pH 4-7 and pH 6-9, about 900 and 600 protein spots were detected by silver staining, respectively. For protein identification, proteins spots on micropreparative gels stained with colloidal Coomassie Brilliant Blue G-250 were excised, digested in-gel with trypsin, and analyzed by peptide mass fingerprinting with delayed extraction-matrix assisted laser desorption/ionization-mass spectrometry (DE-MALDI-MS). In all, 243 protein spots (168 spots in acidic map and 75 spots in basic map) corresponding to 136 different proteins were identified. Besides these principal maps, overview maps (displayed on pH 3-10 gels) for total homogenate and soluble fraction, are also presented with some identifications mapped on them. Based on the 2-DE maps presented in this study, a 2-DE database for human stomach tissue proteome has been constructed and is available at http://proteome.gsnu.ac.kr/DB/2DPAGE/Stomach/. The 2-DE maps and the database resulting from this study will serve important resources for subsequent proteomic studies for analyzing the normal protein variability in healthy tissues and specific protein variations in diseased tissues.     

Two-dimensional Blue Native/sodium dodecyl sulfate gel electrophoresis for analysis of multimeric proteins in platelets

Two-dimensional (2-D) Blue Native/SDS gel electrophoresis combines a first-dimensional separation of monomeric and multimeric proteins in their native state with a second denaturing dimension. These high-resolution 2-D gels aim at identifying multiprotein complexes with respect to their subunit composition. We applied this method for the first time to analyze two human platelet subproteomes: the cytosolic and the microsomal membrane protein fraction. Solubilization of platelet membrane proteins was achieved with the nondenaturing detergent n-dodecyl-beta-D-maltoside. To validate native solubilization conditions, we demonstrated the correct assembly of the Na,K-ATPase, a functional multimeric transmembrane protein, when expressed in Xenopus oocytes. We identified 63 platelet proteins after in-gel tryptic digestion of 58 selected protein spots and liquid chromatography-coupled tandem mass spectrometry. Nine proteins were detected for the first time in platelets by a proteomic approach. We also show that this technology efficiently resolves several known membrane and cytosolic multiprotein complexes. Blue Native/SDS gel electrophoresis is thus a valuable procedure to analyze specific platelet subproteomes, like the membrane(-bound) protein fraction, by mass spectrometry and immunoblotting and could be relevant for the study of protein-protein interactions generated following platelet activation.     

Thin-layer chromatography-densitometry and liquid chromatography analysis of alkaloids in leaves of Papaver somniferum under stress conditions

The effect of stress conditions on the concentrations of secondary metabolites were examined during various developmental stages of Papaver somniferum plants. P. somniferum plants were grown in laboratory conditions (Budakalász). The experiment consisted of 22 treatments. Significantly different alkaloid contents can be observed under different stress conditions. In general, the alkaloid contents of plants are very low; therefore, a highly sensitive and reliable method has to be developed for analysis. The amount of alkaloids was measured by 2 separation and detection techniques. Accuracy of the thin-layer chromatography method for quantitative analysis is limited. Without purification of samples the background is too noisy. Column liquid chromatography is a sensitive and relatively inexpensive method that allows precise quantitative determination of the alkaloid content.     

The mitochondrial proteins of the neuroblastoma cell line IMR-32

Mitochondrial proteins exert important functions in biological pathways, particularly they are involved in apoptotic processes. We applied proteomics technologies to analyze the mitochondrial proteins of the neuroblastoma cell line IMR-32, which is often used in apoptosis studies. The proteins were analyzed by two-dimensional (2-D) electrophoresis followed by matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS). 185 different gene products were identified, of which approximately 55% were enzymes with a broad spectrum of catalytic activities. Sixteen proteins were detected only in this preparation, the others have been detected in two or more protein samples analyzed by MS in our laboratory. The 16 unique gene products were represented by one spot each, whereas most of the frequently detected proteins were represented by multiple spots. In average, approximately 5-10 spots corresponded to one gene product. For two thirds of the proteins identified, an annotation exists in the SWISS-PROT database about their subcellular location. They are mainly described as mitochondrial, 8 as endoplasmic reticulum, 3 as peroxisomal and only 12 low-abundance proteins are described as cytosolic proteins. The list includes about 30 unknown, hypothetical or poorly described gene products. Some of them are represented by strong spots and the present study shows that they are indeed expressed and are localized in the mitochondria.     

基于气相色谱-质谱和网络药理学探讨紫斑罂粟壳挥发油镇咳祛痰平喘的作用机制

为研究紫斑罂粟壳挥发油镇咳化痰平喘的活性成分及作用机制,采用气相色谱-质谱（GC-MS)联用法分析罂粟壳挥发油成分,并结合Pubchem和Swiss Target Prediction数据库筛选活性成分靶点. 其中,在GeneCards数据库中检索镇咳、祛痰、平喘相关的靶点,利用在线Venn取交集基因,Cytoscap 3.7.1软件构建成分-靶点-疾病网络图筛选关键成分,String数据库构建蛋白互作网络筛选核心作用靶点,DAVID数据库进行GO功能和KEGG通路富集分析. 结果表明,GC-MS鉴别出紫斑罂粟壳挥发油中28个化学成分,虚拟筛选获得20个活性成分对应的259个靶点. 网络药理学预测紫斑罂粟壳挥发油通过肿瘤坏死因子（TNF）、磷酸化蛋白激酶（AKT1）、SRC蛋白激酶（SRC）、表皮生长因子受体（EGFR）和丝裂原活化蛋白激酶 1（MAPK1）等关键靶点,进而协同调控肿瘤通路,神经配体-受体相互作用、PI3K-Akt信号通路等多条信号通路发挥镇咳祛痰、平喘的治疗作用. 研究为后续试验研究罂粟壳挥发油的药效物质及作用机制提供参考.     

Analysis of the mouse proteome. (I) Brain proteins: separation by two-dimensional electrophoresis and identification by mass spectrometry and genetic variation

The total protein of the mouse brain was fractionated into three fractions, supernatant, pellet extract and rest pellet suspension, by a procedure that avoids any loss of groups or classes of proteins. The supernatant proteins were resolved to a maximum by large-gel two-dimensional electrophoresis. Two-dimensional patterns from ten individual mice of the commonly used inbred strain C57BL/6 (species: Mus musculus) were prepared. The master pattern was subjected to densitometry, computer-assisted image analysis and treatment with our spot detection program. The resulting two-dimensional pattern, a standard pattern for mouse brain supernatant proteins, was divided into 40 squares, calibrated, and specified by providing each spot with a number. The complete pattern and each of the 40 squares are shown in our homepage (http://www.charite.de/ humangenetik). The standard pattern comprises 8767 protein spots. To identify the proteins known so far in the brain fraction investigated, a first set of 200 spots was analyzed by matrix-assisted laser desorption/ionization - mass spectrometry (MALDI-MS) after in-gel digestion. By screening protein databases 115 spots were identified; by extending the analysis to selected, genetically variant protein spots, 166 spots (including some spot series) were identified in total. This number was increased to 331 by adding protein spots identified indirectly by a genetic approach. By comparing the two-dimensional patterns from C57BL/6 mice with those of another mouse species (Mus spretus), more than 1000 genetically variant spots were detected. The genetic analysis allowed us to recognize spot families, i.e., protein spots that represent the same protein but that are post-translationally modified. If some members of the family were identified, the whole family was considered as being identified. Spot families were investigated in more detail, and interpreted as the result of protein modification or degradation. Genetic analysis led to the interesting finding that the size of spot families, i.e., the extent of modification or degradation of a protein, can be genetically determined. The investigation presented is a first step towards a systematic analysis of the proteome of the mouse. Proteome analysis was shown to become more efficient, and, at the same time, linked to the genome, by combining protein analytical and genetic methods.     

Initial Proteome Analysis of Caffeine-Induced Proteins in <Emphasis Type="Italic">Aspergillus tamarii</Emphasis> Using Two-Dimensional Fluorescence Difference Gel Electrophoresis

Caffeine is toxic to most microorganisms. However, some filamentous fungi, such as Aspergillus tamarii, are able to metabolize this alkaloid when fed caffeine as the sole nitrogen source. The aim of the present work was to identify intracellular A. tamarii proteins, regulated by caffeine, using fluorescence difference two-dimensional gel electrophoresis. Specific proteins from two culture media of A. tamarii grown either on ammonium sulfate or caffeine as the sole nitrogen source were analysed by mass spectrometry. Thirteen out of a total of 85 differentially expressed spots were identified after database search. Identified up-regulated proteins include phosphoglycerate kinase, malate dehydrogenase, dyp-type peroxidase family protein, heat shock protein, Cu, Zn superoxidase dismutase and xanthine dehydrogenase. Some of the proteins identified in this study are involved in the caffeine degradation pathway as well as in stress response, suggesting that stress proteins could be involved in caffeine metabolism in filamentous fungi.     

Assignment of human plasma polypeptides on a nondenaturing 2-D gel using MALDI-MS and PMF and comparisons with the results of intact protein mapping

Human plasma proteins were separated by 2-DE under nondenaturing conditions followed by the assignment of the CBB-stained spots using MALDI-MS and PMF, aiming to correlate the information of intact proteins with that of constituent polypeptides. A microgel system was employed to facilitate the analysis. Totally 157 spots on a nondenaturing micro-2-DE gel were numbered, the spots were excised, the proteins in the gel pieces were subjected to in-gel digestion with trypsin followed by polypeptide analysis using MALDI-MS and PMF. Two PMF algorithms, MASCOT (with Swiss-Prot database) and ProFound (with NCBInr database) were employed. A total of 153 spots out of the 157 provided significant match (p <0.05) with polypeptides in databases. Eighty spots were assigned to contain multiple (2-4) polypeptides, suggesting (i) noncovalent interaction between proteins/polypeptides, (ii) disulfide bonding of polypeptides, or (iii) overlapping of the protein locations on the gel. The results of polypeptide assignment coincided very well with the results of protein mapping previously reported, in which 33 plasma proteins were identified using blotting-immunochemical staining (Manabe, T., Takahashi, Y., Higuchi, N., Okuyama, T., Electrophoresis 1985, 6, 462-467). Further, 19 polypeptides in 25 spots were newly assigned. These results demonstrate that the techniques of MALDI-MS and PMF can be applied for analysis of proteins separated on nondenaturing 2-DE gels, providing information on their polypeptide structure. The integrated information on proteins and polypeptides would help the comprehensive understanding on the functions of complex protein systems.     

Proteomic study of the peripheral proteins from thylakoid membranes of the cyanobacterium Synechocystis sp. PCC 6803

Thylakoid membranes of cyanobacteria and plants contain enzymes that function in diverse metabolic reactions. Many of these enzymes and regulatory proteins are associated with the membranes as peripheral proteins. To identify these proteins, we separated and identified the peripheral proteins of thylakoid membranes of the cyanobacterium Synechocystis sp. PCC 6803. Trichloroacetic acid (TCA)-acetone extraction was used to enrich samples with peripheral proteins and to remove integral membrane proteins. The proteins were separated by two-dimensional electrophoresis (2-DE) and identified by peptide mass fingerprinting. More than 200 proteins were detected on the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel that was stained with colloidal Coomassie blue. We analyzed 116 spots by peptide mass fingerprinting and identified 78 spots that were derived from 51 genes. Some proteins were found in multiple spots, indicating differential modifications resulting in charge differences. Therefore, a significant fraction of the peripheral proteins in thylakoid membranes is modified post-translationally. In our analysis, products of 17 hypothetical genes could be identified in the peripheral protein fraction. Therefore, proteomic analysis is a powerful tool to identify location of the products of hypothetical genes and to characterize complexity in gene expression due to post-translational modifications.     

Comprehensive proteome analysis of mouse liver by ampholyte-free liquid-phase isoelectric focusing

In this study, ampholyte-free liquid-phase IEF (LIEF) was combined with narrow pH range 2-DE and SDS-PAGE RP-HPLC for comprehensive analysis of mouse liver proteome. Because LIEF prefractionation was able to reduce the complexity of the sample and enhance the loading capacity of IEF strips, the number of visible protein spots on subsequent 2-DE gels was significantly increased. A total of 6271 protein spots were detected after integrating five narrow pH range 2-DE gels following LIEF prefractionation into a single virtual 2-DE gel. Furthermore, the pH 3-5 LIEF fraction and the unfractionated sample were separated by pH 3-6 2-DE and identified by MALDI-TOF/TOF MS, respectively. In parallel, the pH 3-5 LIEF fraction was also analyzed by SDS-PAGE RP-HPLC MS/MS. LIEF-2-DE and LIEF-HPLC could obviously improve the separation efficiency and the confidence of protein identification, which identified a higher number of low-abundance proteins and proteins with extreme physicochemical characteristics or post-translational modifications compared to conventional 2-DE method. Furthermore, there were 207 proteins newly identified in mouse liver in comparison with previously reported large-scale datasets. It was observed that the combination of LIEF-2-DE and LIEF-HPLC was effective in promoting MS-based liver proteome profiling and could be applied on similar complex tissue samples.     

Identification of 2D-gel proteins: a comparison of MALDI/TOF peptide mass mapping to mu LC-ESI tandem mass spectrometry

A comparative analysis of protein identification for a total of 162 protein spots separated by two-dimensional gel electrophoresis from two fully sequenced archaea, Methanococcus jannaschii and Pyrococcus furiosus, using MALDI-TOF peptide mass mapping (PMM) and mu LC-MS/MS is presented. 100% of the gel spots analyzed were successfully matched to the predicted proteins in the two corresponding open reading frame databases by mu LC-MS/MS while 97% of them were identified by MALDI-TOF PMM. The high success rate from the PMM resulted from sample desalting/concentrating with ZipTip(C18) and optimization of several PMM search parameters including a 25 ppm average mass tolerance and the application of two different protein molecular weight search windows. By using this strategy, low-molecular weight (<23 kDa) proteins could be identified unambiguously with less than 5 peptide matches. Nine percent of spots were identified as containing multiple proteins. By using mu LC-MS/MS, 50% of the spots analyzed were identified as containing multiple proteins. mu LC-MS/MS demonstrated better protein sequence coverage than MALDI-TOF PMM over the entire mass range of proteins identified. MALDI-TOF and PMM produced unique peptide molecular weight matches that were not identified by mu LC-MS/MS. By incorporating amino acid sequence modifications into database searches, combined sequence coverage obtained from these two complimentary ionization methods exceeded 50% for approximately 70% of the 162 spots analyzed. This improved sequence coverage in combination with enzymatic digestions of different specificity is proposed as a method for analysis of post-translational modification from 2D-gel separated proteins.     

Determination of alkaloids in capsules, milk and ethanolic extracts of poppy (Papaver somniferum L.) by ATR-FT-IR and FT-Raman spectroscopy

Fourier transform (FT) infrared spectroscopy using a diamond composite ATR crystal and NIR-FT-Raman spectroscopy techniques were applied for the simultaneous identification and quantification of the most important alkaloids in poppy capsules. Most of the characteristic Raman signals of the alkaloids can be identified in poppy milk isolated from unripe capsules. But also poppy extracts present specific bands relating clearly to the alkaloid fraction. Raman spectra obtained by excitation with a Nd:YAG laser at 1064 nm show no disturbing fluorescence effects; therefore the plant tissue can be recorded without any special preparation. The used diamond ATR technique allows to measure very small sample amounts (5-10 microL or 2-5 mg) without the necessity to perform time-consuming pre-treatments. When applying cluster analysis a reliable discrimination of "low-alkaloid" and "high-alkaloid" poppy single-plants can be easily achieved. The examples presented in this study provide clear evidence of the benefits of Raman and ATR-IR spectroscopy in efficient quality control, forensic analysis and high-throughput evaluation of poppy breeding material.     

Distribution of Arsenic Species in Different Leaf Fractions – An Evaluation of the Biochemical Deposition of Arsenic in Plant Cells

This paper describes an approach to the determination of arsenic species bonding with proteins or low-molecular peptides by separation of leaf proteins and protein precursors into three fractions and analysis of arsenic species associated to these fractions. Plants irrigated with arsenite contained not only arsenite but also arsenate and dimethylarsinate. In plants treated with arsenate, the major component was arsenite in the water-soluble fraction containing soluble protein and non-protein (F II) and in the acid-soluble non-protein fraction (F IV). Concentrations of 43 mg kg⁻¹ (As(V)-treatment) and 18 mg kg⁻¹ (As(III)-treatment) could be analyzed in the water-insoluble structure protein fraction F I (56 ± 15% of the total mass). Based on the concentration of arsenic species in all fractions, conclusions are drawn over the fixation of arsenic in the fraction of insoluble structure proteins, in the fraction of soluble cytosolic proteins as well as the fraction of amino acids.     

Proteome analysis of mouse brain: two-dimensional electrophoresis profiles of tissue proteins during the course of aging

Mouse brain proteins were isolated from five regions (cerebellum, cerebral cortex, hippocampus, striatum, and cervical spinal cord) at five ages from the 10th week to the 24th month, and separated by two-dimensional gel electrophoresis (2-DE). 2-DE was carried out with an immobilized pH gradient bar in the first dimension, and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the second dimension. Over one thousand protein spots were visualized by silver staining and quantified by image processing. In the analyses, 58 protein spots were distinguishable among the above five brain regions, and 17 proteins were shown to be varied in quantity in the course of aging. Partial amino-terminal sequences and/or internal sequences for a total of 301 protein spots were analyzed. One hundred and eighty proteins appeared to have blocked N-termini and 122 proteins were identified. Twenty-seven new proteins were identified by sequence homology search. A mouse brain proteome database was constructed, which consists of the 2-DE map images and the respective spot data files with 15 related references.     

Characterisation of the rubber elongation factor from ammoniated latex by electrophoresis and mass spectrometry

Rubber elongation factor (REF) is considered as one of the major allergens present in latex. An extraction and purification protocol for preparation of REF standards has been modified. A protein fraction was extracted from ammoniated latex sap and purified by gel filtration chromatography. The purified and concentrated proteins were separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis into two major bands. These bands were further characterised by matrix-assisted laser desorption/ionisation time-of-flight and nano-electrospray ionization mass spectrometry. REF and a truncated form could be ascertained by the mass and fragmentation pattern of the tryptic peptides. In the faster migrating band an additional peptide could be identified. This peptide is also present in Hevb3 and a Mr 27000 latex allergen. Our findings indicate that conventional REF preparations as standards may contain additional allergenic proteins.