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At least 199 records · Page 11Linked to original sources
PubMed2006

Proteomic analysis of a meningococcal outer membrane vesicle vaccine prepared from the group B strain NZ98/254.

In the absence of a suitable carbohydrate-based vaccine, outer membrane vesicle (OMV) vaccines have been used to disrupt outbreaks of serogroup B meningococcal disease for more than 20 years. Proteomic technology provides physical methods with the potential to assess the composition and consistency of these complex vaccines. 2-DE, combined with MS, were used to generate a proteome map of an OMV vaccine, developed to disrupt a long-running outbreak of group B disease in New Zealand. Seventy four spots from the protein map were identified including the outer membrane protein (OMP) antigens: PorA, PorB, RmpM and OpcA. Protein identification indicates that, in addition to OMPs, OMV vaccines contain periplasmic, membrane-associated and cytoplasmic proteins. 2-D-DIGE technology highlighted differences between preclinical development batches of vaccines from two different manufacturers.

Antigens, Bacterial↗
PubMed2005

Development of a sample preparation method for fungal proteomics.

Since filamentous fungi including basidiomycetous fungi possess an exceptionally robust cell wall as in microorganisms, effective extraction of intracellular proteins is a key step for fungal proteomic studies. To overcome the experimental obstacle caused by cell walls, we utilized fungal protoplasts, prepared from the brown-rot basidiomycete, Tyromyces palustris. The amount and quality of proteins extracted from the protoplast cells were much higher than that from the mycelial cells. Quantitative comparisons of proteome maps prepared from mycelial and protoplast cells indicated protein spots with a wider range of molecular weights and pIs in the protoplast sample. Furthermore, no streaking or tailing was observed in the protoplasts, suggesting that effective extraction of intracellular proteins from protoplasts might help suppress degradation of proteins during this process. In addition to the efficiency of protein extraction, simple and efficient subcellular fractionation was also achieved using protoplast cells.

Basidiomycota↗
PubMedDate not supplied

[Study of the mouse liver microsomes by the methods of proteome analysis].

Proteome maps of microsomes and their ghosts (i.e. membranes purified from "ballast" proteins) were obtained using highly purified mouse liver microsomes. Comparative analysis of protein composition of ghosts without and after the induction with phenobarbital (cytochromes P450 inducer) by using 1D- and 2D-electrophoresis and MALDI-TOF-mass-spectrometry revealed more than 30 new proteins, in the course of induction in the 45-60 kDa range (corresponding to the mol. weights of cytochromes P450). In the 17 kDa range (corresponding to the mol. wt. of cytochrome b5) there were 4 additional protein stains about 20 proteins disappeared over the entire electrophoregram). Separation of microsomal ghosts proteins by 1D electrophoresis followed by mass-spectrometric analysis allowed to identify cytochromes P450. The present investigation demonstrates the efficiency of different proteomic methods combination (1D- and 2D-electrophoresis, mass-spectrometry, bioinformatics and determination of the enzyme activities) for cytochromes P450 identification and elucidation of their functioning in different animal tissues and then extrapolating this approach to humans.

Alkylation↗
PubMed2001

Protein expression analysis: from "tip of the iceberg" to a global method.

In this review I will describe the advances that have recently been made in "traditional" two-dimensional gel based protein expression analysis. A major jump has been made toward the automation of gel image analysis and comparison, one of the major bottlenecks in the analysis chain as well as the automation of spot excision and preparation for mass spectrometric analysis. Currently the gel-based "proteome mapping" approach is highly effective and 300 gels and over 10,000 spots a week can be analysed. Very recently, viable alternatives to the use of two-dimensional gel electrophoresis have emerged and these approaches are discussed here. In combination with the recently developed stable isotopic tagging methods for peptide quantitation and new mass spectrometers, this emerging technology will be a rapid and highly effective alternative to gel-based methods with few of the latter's shortcomings.

Electrophoresis, Gel, Two-Dimensional↗
PubMed2005

Parasite genomes.

The availability of genome sequences and the associated transcriptome and proteome mapping projects has revolutionised research in the field of parasitology. As more parasite species are sequenced, comparative and phylogenetic comparisons are improving the quality of gene prediction and annotation. Genome sequences of parasites are also providing important data sets for understanding parasite biology and identifying new vaccine candidates and drug targets. We review some of the preliminary conclusions from examination of parasite genome sequences and discuss some of the bioinformatics approaches taken in this analysis.

Animals↗
PubMed2006

Constitutive and inducible stress proteins dominate the proteome of the murine inner medullary collecting duct-3 (mIMCD3) cell line.

A proteome map of the most abundant proteins in the murine inner medullary collecting duct (mIMCD3) cell line was generated by 2-dimensional gel electrophoresis (2D-GE) combined with MALDI-TOF/TOF mass spectrometry. The 2-D model map identifies 77 distinct constitutive proteins and a total of 86 spots including isoforms. Protein identification was based on both peptide mass fingerprinting (MS) and peptide fragmentation (MS/MS) data. High confidence Mascot scores were obtained in the database search, due to the high quality and the number of MS/MS spectra which provided matching sequence information to the database. A functional classification of the identified proteins showed that a high proportion were stress proteins, such as heat shock proteins and proteins with anti-oxidant activity. Other proteins identified were involved in cytoskeletal maintenance, metabolism and energy generation, as well as in translation, transcription, RNA processing and other cell cycle processes. Exposure of the mIMCD3 cells to hyperosmotic stress using 600 mOsmol/kg NaCl or Urea or 700 mOsmol/kg NaCl-Urea (50:50) resulted in the greatest proteome upregulation in 700 mosM NaCl-Urea and the greatest downregulation in 600 mosM NaCl. Several proteins with molecular chaperone function were induced, such as alpha-B crystallin, two Hsp70 isoforms, the osmotic stress protein (Osp94), as well as aldose reductase. Additional isoforms of the translation elongation factors Eef2 and Eef1a1 were induced. Characterization of the phosphoproteome of mIMCD3 cells with a phosphoprotein-specific stain showed a significant proportion of the proteome was phosphorylated. Additionally, exposure of mIMCD3 cells to 600 mOsmol/kg NaCl hyperosmotic stress resulted in a 1.8-fold higher phosphorylation level of the most acidic isoform of the heat shock protein Hsp27 compared to its phosphorylation level under iso-osmotic conditions.

Aldehyde Reductase↗
PubMed2005

Use of reverse phase protein microarrays and reference standard development for molecular network analysis of metastatic ovarian carcinoma.

Cancer can be defined as a deregulation or hyperactivity in the ongoing network of intracellular and extracellular signaling events. Reverse phase protein microarray technology may offer a new opportunity to measure and profile these signaling pathways, providing data on post-translational phosphorylation events not obtainable by gene microarray analysis. Treatment of ovarian epithelial carcinoma almost always takes place in a metastatic setting since unfortunately the disease is often not detected until later stages. Thus, in addition to elucidation of the molecular network within a tumor specimen, critical questions are to what extent do signaling changes occur upon metastasis and are there common pathway elements that arise in the metastatic microenvironment. For individualized combinatorial therapy, ideal therapeutic selection based on proteomic mapping of phosphorylation end points may require evaluation of the patient's metastatic tissue. Extending these findings to the bedside will require the development of optimized protocols and reference standards. We have developed a reference standard based on a mixture of phosphorylated peptides to begin to address this challenge.

Carcinoma↗
PubMed2002

The proteome of the bacterium Mycoplasma pneumoniae: comparing predicted open reading frames to identified gene products.

An existing proteome map of the bacterium Mycoplasma pneumoniae comprising proteins from 224 genes was extended to 305 genes. This corresponds to about 44% of the 688 proposed genome sequence derived open reading frames (ORFs). The newly assigned gene products were enriched, separated by one-dimensional or two-dimensional (2-D) gel electrophoresis and identified by mass spectrometry. The enrichment procedures included differential centrifugation, anion and cation exchange chromatography, affinity chromatography with heparin as a ligand and isolation of biotinylated proteins by binding to immobilized streptavidin. A comparative analysis of the identified proteins from 305 genes with the as yet unverified 383 ORFs concerning isoelectric point, molecular weight and number of transmembrane segments revealed that proteins with more than three predicted transmembrane segments and an isoelectric point above 10.5 are most likely not to be separated by 2-D gel electrophoresis. The mutual benefits of genomics and proteomics were shown by the identification of a todate unannotated 128 amino acid long protein.

Amino Acid Sequence↗
PubMed2006

Identification of developmentally-regulated proteins in Leishmania panamensis by proteome profiling of promastigotes and axenic amastigotes.

We have employed proteomics to identify proteins upregulated in the amastigote life-stage of Leishmaniapanamensis, using axenically-differentiated forms as models of authentic intracellular parasites. Resolution of the soluble proteomes of axenic amastigotes and promastigotes by two-dimensional electrophoresis (2DE) in the neutral pI range (5-7) revealed equivalent numbers of protein spots in both life-stages (644-682 using Coomassie Blue and 851-863 by silver staining). Although representing a relatively low proportion (8.1-10.8%) of the predicted 8000 gene products of Leishmania, these proteome maps enabled the reproducible detection of 75 differentially-regulated protein spots in amastigotes, comprising 24 spots "uniquely" expressed in this life-stage and 51 over-expressed by 1.2-5.7-fold compared to promastigotes. Of the 11 amastigote-specific spots analysed by mass spectrometry (MS), 5 yielded peptide sequences with no orthologues in Leishmania major, and the remaining 6 were identified as 7 distinct proteins (some of which were truncated isoforms) representing several functional classes: carbohydrate/energy metabolism (fructose 1,6-bisphosphate aldolase, glucose 6-phosphate dehydrogenase, pyruvate dehydrogenase), stress response (heat shock protein [HSP] 83), cell membrane/cytoskeleton (beta-tubulin), amino acid metabolism (cysteine synthase) and cell-cycle (ran-binding protein). Four additional over-expressed spots were tentatively identified as HSPs 60 and 70 and HSP 70-related proteins -1 and -4 by positional analogy with these landmark proteins in the Leishmania guyanensis proteome. Our data demonstrate the feasibility of proteomics as an approach to identify novel developmentally-regulated proteins linked to Leishmania differentiation and intracellular survival, while simultaneously pinpointing therapeutic targets. In particular, the amastigote-specific expression of cysteine synthase underlines the importance of de novo cysteine synthesis both as a potential parasite virulence factor and as a major metabolic difference from mammalian host cells.

Amino Acid Sequence↗
PubMed2004

Selectivity of protein carbonylation in the apoptotic response to oxidative stress associated with photodynamic therapy: a cell biochemical and proteomic investigation.

We previously reported that photodynamic therapy (PDT) using Purpurin-18 (Pu-18) induces apoptosis in HL60 cells. Using flow cytometry, two-dimensional electrophoresis coupled with immunodetection of carbonylated proteins and mass spectrometry, we now show that PDT-induced apoptosis is associated with increased reactive oxygen species generation, glutathione depletion, changes in mitochondrial transmembrane potential, simultaneous downregulation of mitofilin and carbonylation of specific proteins: glucose-regulated protein-78, heat-shock protein 60, heat-shock protein cognate 71, phosphate disulphide isomerase, calreticulin, beta-actin, tubulin-alpha-1-chain and enolase-alpha. Interestingly, all carbonylated proteins except calreticulin and enolase-alpha showed a pI shift in the proteome maps. Our results suggest that PDT with Pu-18 perturbs the normal redox balance and shifts HL60 cells into a state of oxidative stress, which systematically induces the carbonylation of specific chaperones. As these proteins normally produce a prosurvival signal during oxidative stress, we hypothesize that their carbonylation represents a signalling mechanism for apoptosis induced by PDT.

Actins↗
PubMedDate not supplied

Method for identification and quantitative analysis of protein lysine methylation using matrix-assisted laser desorption/ionization--time-of-flight mass spectrometry and amino acid analysis.

Protein methylation is a post-translational modification that might have important functional roles in cell regulation. We present a new technique with sufficient sensitivity (sub-pmol level) for analysis of methylation of proteins in abundances typically found on proteome maps produced by two-dimensional (2-D) gel electrophoresis. The method involves the identification and quantitation of lysine (Lys) methylation using Fmoc (9-fluorenylmethyl chloroformate)-based amino acid analysis (AAA). Tri- and monomethyl-Lys were baseline-separated from other amino acids using a modified buffer system. Trimethyl-Lys was quantitatively recovered after acid hydrolysis and AAA of two known methylated proteins - yeast cytochome c and human calmodulin. The methylated peptides from tryptic digestion of those two proteins were identified by high sensitivity matrix-assisted laser desorption/ionization - time-of-flight (MALDI-TOF) mass spectrometry (MS). An automated mass-screening approach is proposed for the study of various post-translational modifications to understand the distribution of those protein isoforms separated by two-dimensional polyacrylamide gel electrophoresis. It is concluded that the combination of AAA and MALDI-TOF-MS provides a high sensitivity quantitative tool for the analysis of protein post-translational methylation in the context of proteome studies.

Amino Acid Sequence↗
PubMed2004

Comparative analysis of different typing methods for Helicobacter pylori clinical isolates.

The goal of the present work was to compare different techniques of molecular typing using as an example clinical isolates of Helicobacter pylori obtained from patients in different regions of Russia. DNA-macroarray genome scanning using individual genes was employed to set up our basic classification of isolates that did or did not contain pathogenicity islands. In parallel, DNA of the same isolates was used in the conventional random amplified polymorphic DNA (RAPD) PCR analysis, and the isolates were also genotyped (cagA, vacA, iceA, and babA status) and their proteomic maps were obtained by means of unidimensional SDS polyacrylamide gel electrophoresis (1D-SDS-PAGE) coupled with identification using peptide mass fingerprinting by MALDI-TOF mass spectrometry. A statistically significant correlation (coefficient of correlation r = 0.25, p = 0.005) was observed between the results of genome scanning and 1D-SDS-PAGE. No correlation was found between RAPD-PCR typing and genome scanning.

Bacterial Typing Techniques↗
PubMed2000

Proteome analysis of differentially displayed proteins as a tool for the investigation of symbiosis.

Two-dimensional gel electrophoresis was used to identify differentially displayed proteins expressed during the symbiotic interaction between the bacterium Sinorhizobium meliloti strain 1021 and the legume Melilotus alba (white sweetclover). Our aim was to characterize novel symbiosis proteins and to determine how the two symbiotic partners alter their respective metabolisms as part of the interaction, by identifying gene products that are differentially present between the symbiotic and non-symbiotic states. Proteome maps from control M. alba roots, wild-type nodules, cultured S. meliloti, and S. meliloti bacteroids were generated and compared. Over 250 proteins were induced or up-regulated in the nodule, compared with the root, and over 350 proteins were down-regulated in the bacteroid form of the rhizobia, compared with cultured cells. N-terminal amino acid sequencing and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry peptide mass fingerprint analysis, in conjunction with data base searching, were used to assign putative identity to nearly 100 nodule, bacterial, and bacteroid proteins. These included the previously identified nodule proteins leghemoglobin and NifH as well as proteins involved in carbon and nitrogen metabolism in S. meliloti. Bacteroid cells showed down-regulation of several proteins involved in nitrogen acquisition, including glutamine synthetase, urease, a urea-amide binding protein, and a PII isoform, indicating that the bacteroids were nitrogen proficient. The down-regulation of several enzymes involved in polyhydroxybutyrate synthesis and a cell division protein was also observed. This work shows that proteome analysis will be a useful strategy to link sequence information and functional genomics.

Electrophoresis, Gel, Two-Dimensional↗
PubMed2001

Continuous free-flow electrophoresis separation of cytosolic proteins from the human colon carcinoma cell line LIM 1215: a non two-dimensional gel electrophoresis-based proteome analysis strategy.

The conventional approach for analyzing the protein complement of a genome involves the combination of two-dimensional gel electrophoresis (2-DE) and mass spectrometric based protein identification technologies. While 2-DE is a powerful separation technique, it is severely limited by the insolubility of certain classes of proteins (e.g. hydrophobic membrane proteins), as well as the amount of protein that can be processed. Here, we describe a simple procedure for resolving complex mixtures of proteins that involves a combination of free flow electrophoresis (FFE), a liquid-based isoelectric focussing (IEF) method, and sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Resolved proteins were identified by peptide fragment sequencing using capillary column reversed-phase high performance liquid chromatography (RP-HPLC)/mass spectrometry (MS). An initial demonstration of the method was performed using digitonin/ethylenediaminetetraacetic acid EDTA extracted cytosolic proteins from the human colon carcinoma cell line, LIM 1215. Cytosolic proteins were separated by liquid-based IEF (pH range 3-10) into 96 fractions, and each FFE fraction was further fractionated by SDS-PAGE. Selected protein bands were excised from the SDS-PAGE gel, digested in situ with trypsin, and subsequently identified by on-line RP-HPLC/electrospray-ionization ion trap MS. Our results indicate that FFE is: (i) an extremely powerful liquid-based IEF method for resolving proteins; (ii) not limited by the amount of sample that can be loaded onto the instrument; and (iii) capable of fractionating intact protein complexes (a potentially powerful tool for cell-mapping proteomics). An up-to-date list of cytosolic proteins from the human colorectal carcinoma cell line LIM 1215 can be found in the Joint Protein Structure Laboratory (JPSL) proteome database. This information will provide an invaluable resource for future proteomics-based biological studies of colon cancer. The JPSL proteome database can be accessed through the World Wide Web (WWW) network (http://www.ludwig.edu.au/jpsl/jpslhome.html).

Chromatography, High Pressure Liquid↗
PubMed2006

An initial proteomic analysis of human preterm labor: placental membranes.

Human preterm labor (PL) is the single most significant problem in modern Obstetrics and Gynecology, affecting approximately 10% of pregnancies worldwide, constituting the leading cause of perinatal mortality and morbidity, and contributing significantly to chronic childhood disease. Currently, our molecular understanding of PL remains staggeringly inadequate to reliably diagnose or rationally intervene in PL events; several molecular alterations have been implicated in PL, but these have proven of limited value as diagnostic/prognostic markers. The majority of PL events remain spontaneous and unpredictable: critical care emergencies. Here, we apply functional proteomics to dissect molecular mechanisms of human PL. Human placental tissue was collected in clearly differentiated cases of preterm and term labor. Highly refined two-dimensional gel electrophoresis (2DE) was used for protein separation, coupled with automated differential gel image analysis to compare the resulting proteomic maps. For this initial study, only the most important protein differences were selected for further analysis, that is, proteins that were unique to one sample, and absent from the other, with 100% reproducibility across the sample population. In total, 11 such proteins were identified by tandem mass spectrometry, falling into three distinct functional classes: structural/cytoskeletal components, ER lumenal proteins with enzymatic or chaperone functions, and proteins with anticoagulant properties. These expression changes form the groundwork for further molecular investigation of this devastating medical condition. This approach therefore holds the potential not only to define the underlying molecular components, but also to identify novel diagnostic tools and targets for rational drug intervention.

Anticoagulants↗
PubMed2005

Proteome analysis in hippocampus of mice overexpressing human Cu/Zn-superoxide dismutase 1.

Cu/Zn-superoxide dismutase 1 (SOD1), encoded on chromosome 21, is a key enzyme in metabolism of oxygen free radicals and oxidative stress. Transgenic mice overexpressing human SOD1 (Tg-hSOD1) are useful model for Down syndrome (trisomy 21) and familial amyotrophic lateral sclerosis (ALS). It was shown recently that Tg-hSOD1 mice develop a characteristic set of neurodegenerative changes in hippocampus and we therefore decided to study differential protein expression patterns, constructing a mouse hippocampal proteome map using two-dimensional electrophoresis (2-DE) with in-gel digestion of spots followed by matrix-assisted laser desorption/ionisation-time of flight (MALDI-TOF) identification and quantitatively compared protein profiles between non-transgenic mice, hemizygous and homozygous Tg-hSOD1 mice. In total 1056 spots were analysed, resulting in the identification of 445 polypeptides that were the products of 157 different genes. Among these a series of proteins involved in scaffolding, metabolism, signaling and other functions were deranged. Our findings suggest that overexpressed SOD1 directly or by generating reactive oxygen species may lead to aberrant protein expressional patterns that in turn may lead to or reflect neurodegeneration observed in this animal model.

Animals↗
PubMed2001

Proteome analysis of cultivar-specific interactions between Rhizobium leguminosarum biovar trifolii and subterranean clover cultivar Woogenellup.

Proteome analysis was used to identify proteins that are involved in the early stages of nodulation between the subterranean clover cultivar Woogenellup and the Rhizobium leguminosarum bv. trifolii strains ANU843 and ANU794. Strain ANU843 induces nitrogen-fixing nodules whereas strain ANU794 forms aberrant nodules on the roots of cv. Woogenellup that fail to develop beyond an early stage. Our aim was to identify proteins that might be involved in the early stages of nodulation over a 48 h period and to identify proteins that are differentially displayed during the interactions between the host and the two microbes. Proteome maps from control Woogenellup roots and inoculated roots were generated and compared at 24 and 48 h post inoculation. Over 1500 spots were resolved on all gels. Of the 16 protein spots that were differentally displayed or developmentally regulated, 10 were assigned putative identities. These included an alpha-fucosidase, several ethylene-induced proteins, a Cu/Zn superoxide dismutase, a hypothetical 16.5 kDa protein, tubulin alpha-chain, chaperonin 21 precursor and triosephosphate isomerase. Of the 22 constitutively expressed proteins spots examined, eight spots were assigned putative protein homologies through N-terminal sequencing and included several pathogenesis and stress-related proteins. The result may suggest that ethylene levels are upregulated during the early stages of infection but that this does not result in the induction of common pathogenesis-related proteins. The specific induction of alpha-fucosidase by ANU794 may be important in the nodulation failure phenotype of strain ANU794.

Amino Acid Sequence↗
PubMed2004

Changes in the mitochondrial proteome during the anoxia to air transition in rice focus around cytochrome-containing respiratory complexes.

The ability of rice seedlings to grow from dry seed under anoxia provides a rare opportunity in a multicellular eukaryote to study the stages of mitochondrial biogenesis triggered by oxygen availability. The function and proteome of rice mitochondria synthesized under 6 days of anoxia following 1 day of air adaptation have been compared with mitochondria isolated from 7-day aerobically grown rice seedlings. Rice coleoptiles grown under anoxia, and the mitochondria isolated from them respired very slowly compared with air-adapted and air-grown seedlings. Immunodetection of key mitochondrial protein markers, isoelectric focusing electrophoresis followed by SDS-PAGE to make soluble mitochondria proteome maps, and shotgun sequencing of mitochondrial proteins by liquid chromatography-tandem mass spectrometry all revealed similar patterns of the major function categories of mitochondrial proteins from both anoxic and air-adapted samples. Activity analysis showed respiratory oxidases markedly increased in activity during the air adaptation of seedlings. Blue-native electrophoresis followed by SDS-PAGE of mitochondrial membrane proteins clearly showed the very low abundance of assembled b/c complex and cytochrome c(1) oxidase complex in the mitochondrial membrane in anoxic samples and the dramatic increase in the abundance of these complexes on air adaptation. Total heme content, cytochrome absorbance spectra, and the electron carrier, cytochrome c, also increased markedly on air adaptation. These results likely reflect limited heme synthesis for cytochrome assembly in the absence of oxygen and represent a discrete and reversible blockage of full mitochondrial biogenesis in this anoxia-tolerant species.

Air↗