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.     

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.     

Column chromatographic prefractionation leads to the detection of 543 different gene products in human fetal brain

In a previous publication a large series of proteins were identified in fetal human brain by the use of two-dimensional electrophoresis (2-DE) with subsequent matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) and MALDI-tandem time-of-flight (TOF/TOF) analysis. Further identification of many more different spots by traditional 2-DE without additional step such as narrow immobilized ph gradient (IPG) strips or prefractionation seems unlikely and we therefore decided to separate extracted brain proteins by ion-exchange chromatography using a TSK gel DEAE-5PW column followed by 2-DE of individual fractions and analysis by MALDI-TOF/TOF with LIFT technology in fetal brain of the early second trimester. About 1880 protein spots corresponding to 543 different gene products were identified. These proteins included housekeeping, signaling, cytoskeletal, metabolic, antioxidant, and neuron/synaptosomal specific proteins. Among these, 314 gene products (314/543, 57.8%), which have never been detected in traditional 2-DE of human fetal brain, were observed by this method. This updated map of fetal brain proteins may serve as data base and reference map for fetal brain proteins, and the methodology applied may be used as a valuable analytical tool for the basis of protein expressional studies in health and disease.     

Enrichment of low-copy-number gene products by hydrophobic interaction chromatography

Enrichment of proteins in solution is the goal of a purification process and often a scientific challenge. We investigated the capacity of hydrophobic interaction chromatography to enrich proteins, potential candidates for novel drug targets. The soluble protein fraction of Haemophilus influenzae was fractionated over a TSK Phenyl column and the proteins resolved were analyzed by two-dimensional gel electrophoresis and matrix-assisted laser desorption ionization mass spectrometry. Approximately 150 proteins, bound to the column, were identified, 30 for the first time. Most of the proteins enriched by hydrophobic interaction chromatography were represented by major spots, so that an enrichment of low-copy-number gene products was only partially achieved. The proteins enriched by this chromatographic approach belong to various protein classes, including enzymes, ribosomal proteins and proteins with as yet unknown functions. The results include two-dimensional maps and a list of the proteins enriched by hydrophobic interaction chromatography.     

Two-dimensional gel electrophoresis maps of the proteome and phosphoproteome of primitively cultured rat mesangial cells

Mesangial cells (MC) play an important role in maintaining the structure and function of the glomerulus. The proliferation of MC is a prominent feature of many kinds of glomerular disease. The first reference 2-DE maps of rat mesangial cells (RMC), stained with silver staining or Pro-Q Diamond dye, have been established here to describe the proteome and phosphoproteome of RMC, respectively. A total of 157 selected protein spots, corresponding to 118 unique proteins, have been identified by MALDI-TOF-MS or LC-ESI-IT-MS/MS, in which 37 protein spots representing 28 unique proteins have also been stained with Pro-Q Diamond, indicating that they are in phosphorylated forms. All the identified proteins were bioinformatically annotated in detail according to their physiochemical characteristics, subcellular location, and function. Most of the separated or identified protein spots are distributed in the area of mass 10-70 kDa and pI 5.0-8.0. The identified proteins include mainly cytoplasmic and nuclear proteins and some mitochondrial, endoplasmic reticulum, and membrane proteins. These proteins are classified into different functional groups such as structure and mobility proteins (21.2%), metabolic enzymes (16.9%), protein folding and metabolism proteins (13.6%), signaling proteins (14.4%), heat-shock proteins (7.6%), and other functional proteins (12.7%). While structure and mobility proteins are mostly represented by protein spots with high abundance, signaling proteins are mostly represented by protein spots with relatively low abundance. Such a 2-DE database for RMC, especially with many signaling proteins and phosphoproteins characterized, will provide a valuable resource for comparative proteomics analysis of normal and pathologic conditions affecting MC function or pathologic progress.     

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.     

Complementing genomics with proteomics: the membrane subproteome of Pseudomonas aeruginosa PAO1

With the completion of many genome projects, a shift is now occurring from the acquisition of gene sequence to understanding the role and context of gene products within the genome. The opportunistic pathogen Pseudomonas aeruginosa is one organism for which a genome sequence is now available, including the annotation of open reading frames (ORFs). However, approximately one third of the ORFs are as yet undefined in function. Proteomics can complement genomics, by characterising gene products and their response to a variety of biological and environmental influences. In this study we have established the first two-dimensional gel electrophoresis reference map of proteins from the membrane fraction of P. aeruginosa strain PA01. A total of 189 proteins have been identified and correlated with 104 genes from the P. aeruginosa genome. Annotated membrane proteins could be grouped into three distinct categories: (i) those with functions previously characterised in P. aeruginosa (38%); (ii) those with significant sequence similarity to proteins with assigned function or hypothetical proteins in other organisms (46%); and (iii) those with unknown function (16%). Transmembrane prediction algorithms showed that each identified protein sequence contained at least one membrane-spanning region. Furthermore, the current methodology used to isolate the membrane fraction was shown to be highly specific since no contaminating cytosolic proteins were characterised. Preliminary analysis showed that at least 15 gel spots may be glycosylated in vivo, including three proteins that have not previously been functionally characterised. The reference map of membrane proteins from this organism is now the basis for determining surface molecules associated with antibiotic resistance and efflux, cell-cell signalling and pathogen-host interactions in a variety of P. aeruginosa strains.     

Protein profiling of rat cerebella during development

Protein profiles of developing rat cerebella were analyzed by means of two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS). The analysis of adult rat cerebellum gave rise to a protein map comprising approximately 3000 spots detectable by silver staining following high resolution 2-DE with a pH range of 3-10 and a mass range of 8-100 kDa. To obtain landmarks for comparison of developmental profiles of cerebellar proteins, 100 spots were subjected to peptide mass fingerprinting using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), and 67 spots were assigned on the map. Analysis of profiles of the developing cerebella revealed significant changes in the expression of proteins during development. In most cases the expression levels of proteins increased as the cerebellum matured, while the expression of 42 spots appeared specific or remarkably abundant in the immature cerebellum. Peptide mass fingerprinting of these spots allowed us to identify 29 proteins, which include, in addition to proteins of unknown function, many proteins known to have roles in the development of the central nervous system. These results suggest that the proteomic approach is valuable for mass identification of proteins involved in cerebellar morphogenesis.     

Enrichment of low abundance proteins of Escherichia coli by hydroxyapatite chromatography.

Visualization of low-copy-number gene products is essential for the detection of novel drug targets by differential protein expression studies. We investigated the enrichment of low-abundance proteins of Escherichia coli by hydroxyapatite chromatography. The proteins of the various pools collected from a ceramic hydroxyapatite column were analyzed by two-dimensional electrophoresis and identified by matrix-assisted laser desorption ionization mass spectrometry. Approximately 800 spots corresponding to 296 different proteins were identified in the hydroxyapatite eluate. About 130 proteins that had not been detected in the two-dimensional gels of the total extract were identified. Hydroxyapatite chromatography enriched low-abundance but also major components of the E. coli protein extract. In particular, it enriched many low-molecular-mass proteins, such as cold-shock proteins. The proteins bound to the hydroxyapatite matrix belong to several classes, including enzymes with various catalytic activities, heat- and cold-shock proteins and many hypothetical and novel proteins with yet unknown functions. The results include a list of the proteins enriched by hydroxyapatite chromatography and a two-dimensional map of the enriched proteins. They may be useful in the design of protein purification pathways using master purification steps and in the search for novel drug targets.     

An integrated map of the murine hippocampal proteome based upon five mouse strains

With the advent of proteomics technologies it is possible to simultaneously demonstrate the expression of hundreds of proteins. The information offered by proteomics provides context-based understanding of cellular protein networks and has been proven to be a valuable approach in neuroscience studies. The mouse hippocampus has been a major target of analysis in the search for molecular correlates to neuronal information storage. Although human and rat hippocampal samples have been successfully subjected to proteomic profiling, no elaborate analysis providing the fundamental experimental basis for protein-expression studies in the mouse hippocampus has been carried out as yet. This led us to construct a master map generated from the individual hippocampal proteomes of five different mouse strains. A proteomic approach, based upon 2-DE coupled to MS (MALDI-TOF/TOF) has been chosen in an attempt to establish a comprehensive reference database of proteins expressed in the mouse hippocampus. 469 individual proteins, represented by 1156 spots displaying various functional states of the respective gene products were identified. Proteomic profiling of the hippocampus, a brain region with a pivotal role for neuronal information processing and storage may provide insight into the characteristics of proteins serving this highly sophisticated function.     

大鼠骨髓间充质干细胞分化过程的比较蛋白质组学研究

从蛋白质组学角度分析大鼠骨髓间充质干细胞(MSCs)体外定向分化为心肌细胞过程中蛋白表达情况, 采用二维电泳分离蛋白, 用PDQuest软件分析蛋白表达差异, 并采用质谱(MALDI-TOF-MS)进行鉴定, 得到了54个蛋白点, 对蛋白的生物功能分析表明, 部分蛋白通过不同的信号途径参与了MSCs的分化过程.     

Identification of proteins from Mycobacterium tuberculosis missing in attenuated Mycobacterium bovis BCG strains

A proteome approach, combining high-resolution two-dimensional electrophoresis (2-DE) with mass spectrometry, was used to compare the cellular protein composition of two virulent strains of Mycobacterium tuberculosis with two attenuated strains of Mycobacterium bovis Bacillus Calmette-Guerin (BCG), in order to identify unique proteins of these strains. Emphasis was given to the identification of M. tuberculosis specific proteins, because we consider these proteins to represent putative virulence factors and interesting candidates for vaccination and diagnosis of tuberculosis. The genome of M. tuberculosis strain H37Rv comprises nearly 4000 predicted open reading frames. In contrast, the separation of proteins from whole mycobacterial cells by 2-DE resulted in silver-stained patterns comprising about 1800 distinct protein spots. Amongst these, 96 spots were exclusively detected either in the virulent (56 spots) or in the attenuated (40 spots) mycobacterial strains. Fifty-three of these spots were analyzed by mass spectrometry, of which 41 were identified, including 32 M. tuberculosis specific spots. Twelve M. tuberculosis specific spots were identified as proteins, encoded by genes previously reported to be deleted in M. bovis BCG. The remaining 20 spots unique for M. tuberculosis were identified as proteins encoded by genes that are not known to be missing in M. bovis BCG.     

Identifying the major proteome components of Helicobacter pylori strain 26695

The whole genome sequences of Helicobacter pylori strain 26695 have been reported. Whole cell proteins of H. pylori strain 26695 cells were obtained and analyzed by two-dimensional electrophoresis, using immobilized pH gradient strips. The most abundant proteins were shown in the region of pI 4.0-9.5 with molecular masses from 10 to 100 kDa. Soluble proteins were precipitated by the use of 0-80% saturated solutions of ammonium sulfate. Soluble proteins precipitated by the 0-40% saturations of ammonium sulfate produced similar spot profiles and their abundant protein spots had acidic pI regions. However, a number of soluble proteins precipitated by more than 60% saturation of ammonium sulfate were placed in the alkaline pI regions, compared to those precipitated by 40% saturation. In addition, we have performed an extensive proteome analysis of the strain utilizing peptide MALDI-TOF-MS. Among the 345 protein spots processed, 175 proteins were identified. The identified spots represented 137 genes. One-hundred and fifteen proteins were newly identified in this study, including DNA polymerase III beta-subunit. These results might provide guidance for the enrichment of H. pylori proteins and contribute to construct a master protein map of H. pylori.     

A mouse serum two-dimensional gel map: application to profiling burn injury and infection

With the importance of mouse as a model to study human diseases and the human and rat plasma/serum two-dimensional (2-D) maps being extensively annotated, this study was aimed at constructing a detailed mouse serum 2-D map. Serum proteins from two different inbred strains of mice (BALB/cJ and C57BL/6J) and mice subjected to two different inflammatory stimuli (20% burn injury and lipopolysaccharide (LPS) injection) were separated on overlapping gels covering pH 3-8 and stained with SYPRO Ruby dye. The tryptic peptides from the resolved spots were analyzed by mass spectrometry, leading to the identification of 38 different gene products. With the exception of major urinary proteins found in abundance in male C57BL/6J mice, little strain difference of the mouse serum 2-D was observed. Many proteins detected in the mouse serum 2-D map were not reported in human or rat serum 2-D maps including epidermal growth factor receptor. Three major murine acute-phase proteins (APPs), haptoglobin, serum amyloid A, and serum amyloid P, were highly induced by both inflammatory stimuli. Image analysis shows that the variations of APPs between these two inflammatory models were not uniform although LPS (100 microg/animal) in general was more effective than 20% burn injury in inducing APPs. Serum amyloid A, much more sensitive to endotoxin than burn injury, may represent a sensitive marker to differentiate these two different inflammatory states.     

Combinatorial peptide ligand library plasma treatment: Advantages for accessing low‐abundance proteins

Depletion of major blood proteins is one of the most promising approaches to accessing low‐abundance biomarkers. This study compared the use of combinatorial peptide ligand library (CPLL) and albumin and immunoglobulins (IgGs) depletion technology for accessing these low‐abundance proteins in plasma using 2‐DE in an acidic restricted pH range (4–7). Compared with native plasma, both techniques enlarge the visibility of other proteins than albumin and IgG, but there were marked differences in their composition. An increase of the number of spots was detected compared with native plasma (157 spots) with 427 and 557 spots, respectively, detected with albumin and IgG depletion, and CPLL treatment. We selected 70 spots to be identified by MALDI‐TOF related to their absence in the 2‐D gels from native or albumin and IgG‐depleted plasma. The 42 spots identified corresponded to 24 different proteins, with more than half of the proteins which did not belong to the major plasma proteins. CPLL treatment allowed the accessibility to proteolytic fragments obtained from major plasma proteins. We found a large superiority of the CPLL approach over the albumin and IgG depletion process. These findings show the utility of depleting major blood proteins to be able to access low‐abundance proteins and the potential of CPLL to select and identify candidate biomarkers in clinical studies.     

A high-resolution reference map for cytoplasmic and membrane-associated proteins of Corynebacterium glutamicum

We present a high-resolution reference map for soluble proteins obtained from Corynebacterium glutamicum cells grown in glucose minimal medium. The analysis window covers the pl range from 4-6 and the molecular mass range from 5-100 kDa. Using overlapping narrow immobilized pH gradients for isoelectric focusing, 970 protein spots were detected after second-dimensional separation on SDS-polyacrylamide gels and colloidal Coomassie-staining. By tryptic peptide mass fingerprinting 169 protein spots were identified, representing 152 different proteins including many enzymes involved in central metabolism (18), amino acid biosynthesis (24) and nucleotide biosynthesis (11). Thirty-five of the identified proteins have no known function. A comparison of the observed and the expected physicochemical properties of the identified proteins indicated that nine proteins were covalently modified, since variants with apparently identical molecular mass, but differing pl were detected. The N-termini of eight proteins were determined by post-source decay (PSD) analysis of selected peptides. In addition to the soluble proteins, a map of the membrane-bound proteins within the pl range 4-7 is presented, which contains 660 protein spots, 22 of which were identified, representing 13 different proteins.     

Proteomic Profiling and the Predicted Interactome of Host Proteins in Compatible and Incompatible Interactions Between Soybean and <Emphasis Type="Italic">Fusarium virguliforme</Emphasis>

Sudden death syndrome (SDS) is a complex of two diseases of soybean (Glycine max), caused by the soil borne pathogenic fungus Fusarium virguliforme. The root rot and leaf scorch diseases both result in significant yield losses worldwide. Partial SDS resistance has been demonstrated in multiple soybean cultivars. This study aimed to highlight proteomic changes in soybean roots by identifying proteins which are differentially expressed in near isogenic lines (NILs) contrasting at the Rhg1/Rfs2 locus for partial resistance or susceptibility to SDS. Two-dimensional gel electrophoresis resolved approximately 1000 spots on each gel; 12 spots with a significant (P < 0.05) difference in abundance of 1.5-fold or more were picked, trypsin-digested, and analyzed using quadruple time-of-flight tandem mass spectrometry. Several spots contained more than one protein, so that 18 distinct proteins were identified overall. A functional analysis performed to categorize the proteins depicted that the major pathways altered by fungal infection include disease resistance, stress tolerance, and metabolism. This is the first report which identifies proteins whose abundances are altered in response to fungal infection leading to SDS. The results provide valuable information about SDS resistance in soybean plants, and plant partial resistance responses in general. More importantly, several of the identified proteins could be good candidates for the development of SDS-resistant soybean plants.     

Differential Proteomic Analysis of Neonatal Cardiomyocytes in Response to β-Adrenergic Receptor Stimulation

β-Adrenoceptors(β-ARs) play a critical role in regulating cardiac functions under both physiological and pathological conditions. To further explore the mechanisms through whichβ-ARs perform its actions, proteomic approaches were adopted to study the global protein patterns in cultured neonatal rat cardiomyocytes exposed to isoproterenol(ISO). A modified method, "Mirror Images in One Gel", was used to improve the reproducibility and resolution power of two-dimensional electrophoresis. A 2-DE map with a good reproducibility was obtained in which 1281 ± 70spots were detected and about 1191 e 54 spots were matched, with an average matching rate of 92. 9%. Nine proteins with significant changes were identified by using peptide mass fingerprinting(PMF) data obtained via MALDI-MS.     

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.