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PubMed2006

Proteome profiling in the rat Harderian gland.

The Harderian gland is an orbital gland located behind the ocular bulb in most terrestrial vertebrates probably functioning for production of lipid secretion to protect the eye. We herein present a protein reference database of the rat Harderian gland that may serve as analytical tool for future proteomic work, report lipid and porphyrin handling cascades, address sequence conflicts and report structures that have not been so far described by proteomics methods.

Amino Acid Sequence↗
PubMed2006

High dynamic range characterization of the trauma patient plasma proteome.

Although human plasma represents an attractive sample for disease biomarker discovery, the extreme complexity and large dynamic range in protein concentrations present significant challenges for characterization, candidate biomarker discovery, and validation. Herein we describe a strategy that combines immunoaffinity subtraction and subsequent chemical fractionation based on cysteinyl peptide and N-glycopeptide captures with two-dimensional LC-MS/MS to increase the dynamic range of analysis for plasma. Application of this "divide-and-conquer" strategy to trauma patient plasma significantly improved the overall dynamic range of detection and resulted in confident identification of 22,267 unique peptides from four different peptide populations (cysteinyl peptides, non-cysteinyl peptides, N-glycopeptides, and non-glycopeptides) that covered 3,654 different proteins with 1,494 proteins identified by multiple peptides. Numerous low abundance proteins were identified, exemplified by 78 "classic" cytokines and cytokine receptors and by 136 human cell differentiation molecules. Additionally a total of 2,910 different N-glycopeptides that correspond to 662 N-glycoproteins and 1,553 N-glycosylation sites were identified. A panel of the proteins identified in this study is known to be involved in inflammation and immune responses. This study established an extensive reference protein database for trauma patients that provides a foundation for future high throughput quantitative plasma proteomic studies designed to elucidate the mechanisms that underlie systemic inflammatory responses.

Blood Proteins↗
PubMed2002

Proteomic analysis of normal human urinary proteins isolated by acetone precipitation or ultracentrifugation.

BACKGROUND: Proteomic techniques have recently become available for large-scale protein analysis. The utility of these techniques in identification of urinary proteins is poorly defined. We constructed a proteome map of normal human urine as a reference protein database by using two differential fractionated techniques to isolate the proteins. METHODS: Proteins were isolated from urine obtained from normal human volunteers by acetone precipitation or ultracentrifugation, separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and identified by matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry followed by peptide mass fingerprinting. RESULTS: A total of 67 protein forms of 47 unique proteins were identified, including transporters, adhesion molecules, complement, chaperones, receptors, enzymes, serpins, cell signaling proteins and matrix proteins. Acetone precipitated more acidic and hydrophilic proteins, whereas ultracentrifugation fractionated more basic, hydrophobic, and membrane proteins. Bioinformatic analysis predicted glycosylation to be the most common explanation for multiple forms of the same protein. CONCLUSIONS: Combining two differential isolation techniques magnified protein identification from human urine. Proteomic analysis of urinary proteins is a promising tool to study renal physiology and pathophysiology and to determine biomarkers of renal disease.

Acetone↗
PubMed2005

Proteome analysis of mouse primary astrocytes.

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

Animals↗
PubMed1997

Development of a Haemophilus two-dimensional protein database.

Members of the Haemophilus genus are responsible for various human infections including respiratory infections and meningitis. The complete nucleotide sequence of the Rd strain of Haemophilus influenzae has been reported and represents a valuable resource to investigate gene expression within this bacterial group. We described previously the application of two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) to characterise the proteins of Haemophilus influenzae (Cash et al., Electrophoresis 1995, 16, 135-148). We have extended these data with comparative studies of the proteins from other members of the Haemophilus genus (specifically H. parainfluenzae, H. haemolyticus and H. parahaemolyticus) to identify homologous proteins and, by extension, the genes encoding them, among these bacteria. The proteins extracted from each of these bacterial isolates were compared by coelectrophoresis to the 2-D protein profile of the reference nontypable strain of H. influenzae (HI-64443) used as the basis for the 2-D protein database. A composite reference 2-D protein profile of HI-64443 was derived from three independent analyses of the soluble bacterial proteins. Between 21% and 37% of the HI-64443 proteins from the reference 2-D protein profile comigrated with proteins in the other isolates from the Haemophilus genus. This compared with 62% and 64% comigration when HI-64443 was compared with the Eagan and Rd strains of H. influenzae, respectively. The 2-D protein profile of the Rd strain of H. influenzae was compared to that of HI-64443 by coelectrophoresis; 64% of the proteins detected for the Rd strain comigrated with proteins found for HI-64443 when analysed in parallel. The capacity of 2-D PAGE to investigate global interactions of gene expression was applied to the analysis of superoxide dismutase (SOD) expression in H. influenzae strain Eagan. A "knock-out" mutant in the sodA gene which encodes [Mn]-SOD was characterised with respect to protein synthesis compared to the parental isolate. From these analyses, the primary product of sodA was provisionally identified as a protein with a molecular mass of 25500 Da and an estimated pI of 6.55. Quantitative changes in the expression of two other proteins in the SOD mutant were detected by comparison with the parental isolate. These data are discussed in relation to the development of a 2-D protein database for H. influenzae and related bacteria to investigate genome homologies and gene expression.

Bacterial Proteins↗
PubMed2006

The TIGR Plant Transcript Assemblies database.

The TIGR Plant Transcript Assemblies (TA) database (http://plantta.tigr.org) uses expressed sequences collected from the NCBI GenBank Nucleotide database for the construction of transcript assemblies. The sequences collected include expressed sequence tags (ESTs) and full-length and partial cDNAs, but exclude computationally predicted gene sequences. The TA database includes all plant species for which more than 1000 EST or cDNA sequences are publicly available. The EST and cDNA sequences are first clustered based on an all-versus-all pairwise sequence comparison, followed by the generation of consensus sequences (TAs) from individual clusters. The clustering and assembly procedures use the TGICL tool, Megablast and the CAP3 assembler. The UniProt Reference Clusters (UniRef100) protein database is used as the reference database for the functional annotation of the assemblies. The transcription orientation of each TA is determined based on the orientation of the alignment with the best protein hit. The TA sequences and annotation are available via web interfaces and FTP downloads. Assemblies can be retrieved by a text-based keyword search or a sequence-based BLAST search. The current version of the TA database is Release 2 (July 17, 2006) and includes a total of 215 plant species.

DNA, Complementary↗
PubMed2004

The RESID Database of Protein Modifications as a resource and annotation tool.

The RESID Database of Protein Modifications is a comprehensive collection of annotations and structures for protein modifications and cross-links including pre-, co-, and post-translational modifications. The database provides: systematic and alternate names, atomic formulas and masses, enzymatic activities that generate the modifications, keywords, literature citations, Gene Ontology (GO) cross-references, protein sequence database feature table annotations, structure diagrams, and molecular models. This database is freely accessible on the Internet through resources provided by the European Bioinformatics Institute (http://www.ebi.ac.uk/RESID), and by the National Cancer Institute--Frederick Advanced Biomedical Computing Center (http://www.ncifcrf.gov/RESID). Each RESID Database entry presents a chemically unique modification and shows how that modification is currently annotated in the protein sequence databases, Swiss-Prot and the Protein Information Resource (PIR). The RESID Database provides a table of corresponding equivalent feature annotations that is used in the UniProt project, an international effort to combine the resources of the Swiss-Prot, TrEMBL and PIR. As an annotation tool, the RESID Database is used in standardizing and enhancing modification descriptions in the feature tables of Swiss-Prot entries. As an Internet resource, the RESID Database assists researchers in high-throughput proteomics to search monoisotopic masses and mass differences and identify known and predicted protein modifications.

Databases, Factual↗
PubMed2005

An automated method for the analysis of stable isotope labeling data in proteomics.

An algorithm is presented for the generation of a reliable peptide component peak table from liquid chromatography-mass spectrometry (LC-MS) and subsequent quantitative analysis of stable isotope coded peptide samples. The method uses chemical noise filtering, charge state fitting, and deisotoping toward improved analysis of complex peptide samples. Overlapping peptide signals in mass spectra were deconvoluted by correlation with modeled peptide isotopic peak profiles. Isotopic peak profiles for peptides were generated in silico from a protein database producing reference model distributions. Doublets of heavy and light labeled peak clusters were identified and compared to provide differential quantification of pairs of stable isotope coded peptides. Algorithms were evaluated using peptides from digests of a single protein and a seven-protein mixture that had been differentially coded with stable isotope labeling agents and mixed in known ratios. The experimental results correlated well with known mixing ratios.

Algorithms↗
PubMed1999

BOLD--a biological O-linked glycan database.

Glycans can be O-linked to proteins via the hydroxyl group of serine, threonine, tyrosine, hydroxylysine or hydroxyproline. Sometimes the glycan is O-linked to the hydroxyl group via a phosphodiester bond. The core monosaccharide residue may be N-acetylgalactosamine, N-acetylglucosamine, galactose, glucose, fucose, mannose, xylose or arabinose. These O-linked glycans can remain as a monosaccharide, but often a complex structure is built up by stepwise addition of monosaccharides. Monosaccharides known to be added include galactose, N-acetylglucosamine, fucose, N-acetylneuraminic acid, N-glycolylneuraminic acid and 2-keto-3-deoxynonulosonic acid. O-linked glycans can also contain sulfate and phosphate residues. This leads to the possibility of the existence of numerous O-glycan structures. The biological O-linked database (BOLD) is a relational database that contains information on O-linked glycan structures, their biological sources (with a link to the SWISS-PROT protein database), the references in which the glycan was described (with a link to MEDLINE), and the methods used to determine the glycan structure. The database provides a valuable resource for glycobiology researchers interested in O-linked oligosaccharide structures that have been previously described on proteins from different species and tissues.

Carbohydrate Conformation↗
PubMed2003

RefDB: a database of uniformly referenced protein chemical shifts.

RefDB is a secondary database of reference-corrected protein chemical shifts derived from the BioMagResBank (BMRB). The database was assembled by using a recently developed program (SHIFTX) to predict protein (1)H, (13)C and (15)N chemical shifts from X-ray or NMR coordinate data of previously assigned proteins. The predicted shifts were then compared with the corresponding observed shifts and a variety of statistical evaluations performed. In this way, potential mis-assignments, typographical errors and chemical referencing errors could be identified and, in many cases, corrected. This approach allows for an unbiased, instrument-independent solution to the problem of retrospectively re-referencing published protein chemical shifts. Results from this study indicate that nearly 25% of BMRB entries with (13)C protein assignments and 27% of BMRB entries with (15)N protein assignments required significant chemical shift reference readjustments. Additionally, nearly 40% of protein entries deposited in the BioMagResBank appear to have at least one assignment error. From this study it evident that protein NMR spectroscopists are increasingly adhering to recommended IUPAC (13)C and (15)N chemical shift referencing conventions, however, approximately 20% of newly deposited protein entries in the BMRB are still being incorrectly referenced. This is cause for some concern. However, the utilization of RefDB and its companion programs may help mitigate this ongoing problem. RefDB is updated weekly and the database, along with its associated software, is freely available at http://redpoll.pharmacy.ualberta.ca and the BMRB website.

Animals↗
PubMed2005

NCBI Reference Sequence (RefSeq): a curated non-redundant sequence database of genomes, transcripts and proteins.

The National Center for Biotechnology Information (NCBI) Reference Sequence (RefSeq) database (http://www.ncbi.nlm.nih.gov/RefSeq/) provides a non-redundant collection of sequences representing genomic data, transcripts and proteins. Although the goal is to provide a comprehensive dataset representing the complete sequence information for any given species, the database pragmatically includes sequence data that are currently publicly available in the archival databases. The database incorporates data from over 2400 organisms and includes over one million proteins representing significant taxonomic diversity spanning prokaryotes, eukaryotes and viruses. Nucleotide and protein sequences are explicitly linked, and the sequences are linked to other resources including the NCBI Map Viewer and Gene. Sequences are annotated to include coding regions, conserved domains, variation, references, names, database cross-references, and other features using a combined approach of collaboration and other input from the scientific community, automated annotation, propagation from GenBank and curation by NCBI staff.

Animals↗
PubMed2000

Serum protein immunogenicity: implications for liver xenografting.

The objectives of this study were threefold: (i) assess immunogenicity of donor plasma proteins following hepatic xenotransplantation, (ii) identify potential immunogens, and (iii) consider the implications of antibody formation against these plasma proteins in xenograft survival. We studied liver and heart xenografts in a concordant combination, hamster to rat. All grafts were examined at necropsy for evidence of rat immunoglobulin G (IgG) deposition. Cardiac xenografts were placed in recipients who had, or had not, been sensitized with hamster serum. Hepatic xenografts were placed in naive recipients to see if antibodies to hamster serum proteins could be eluted from the rejecting organ. Sera of immunized rats were examined for the presence of anti-hamster antibodies by immunoelectrophoresis and by Western blotting following sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) separation of hamster serum. Antibodies in sera of immunized rats were compared with those eluted from rejecting livers. Candidate antigens were identified by tandem mass spectrometry, sequence analysis, and reference to protein databases. Results showed that sera of immunized rats recognized a minimum of four different antigens in hamster serum by immunoelectrophoresis, and a minimum of seven by the more sensitive SDS-PAGE Western blot. IgG eluted from rejecting livers bound three of seven candidate antigens recognized by sera of the immunized animals. Sequence analysis searches revealed proteinase inhibitors in each of the three SDS-PAGE bands common to the above samples. All of these candidate proteinase inhibitor immunogens share a common catabolic fate, uptake via the lipoprotein-related protein (LRP/alpha 2-macroglobulin receptor (CD91). Sensitization to hamster serum proteins hastened cardiac xenograft rejection in 30-50% of recipients (depending on sensitization protocol). Vascular deposition of rat IgG occurred in all rejecting xenografts. Antibody binding to proteinase inhibitors could disturb their functional activity and contribute to the pathogenesis of delayed xenograft rejection.

Amino Acid Sequence↗
PubMed2005

Rice proteome analysis: a step toward functional analysis of the rice genome.

The technique of proteome analysis using 2-DE has the power to monitor global changes that occur in the protein complement of tissues and subcellular compartments. In this review, we describe construction of the rice proteome database, the cataloging of rice proteins, and the functional characterization of some of the proteins identified. Initially, proteins extracted from various tissues and organelles were separated by 2-DE and an image analyzer was used to construct a display or reference map of the proteins. The rice proteome database currently contains 23 reference maps based on 2-DE of proteins from different rice tissues and subcellular compartments. These reference maps comprise 13 129 rice proteins, and the amino acid sequences of 5092 of these proteins are entered in the database. Major proteins involved in growth or stress responses have been identified by using a proteomics approach and some of these proteins have unique functions. Furthermore, initial work has also begun on analyzing the phosphoproteome and protein-protein interactions in rice. The information obtained from the rice proteome database will aid in the molecular cloning of rice genes and in predicting the function of unknown proteins.

Calreticulin↗
PubMed2002

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

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

Animals↗
PubMed2001

Differential protein expression in rat trigeminal ganglia during inflammation.

A proteomics approach was used to investigate global protein changes in rat sensory ganglia exposed to pro-inflammatory stimuli. Inflammation was provoked in vivo by injecting selected facial areas with Freunds Complete Adjuvant (FCA), or by stimulating freshly isolated trigeminal ganglia ex vivo with pro-inflammatory mediators (interleukin-1 beta, tumor necrosis factor-alpha, interferon-gamma). Following two-dimensional gel electrophoresis, silver staining and mass spectrometry, a protein reference map and database was generated for rat trigeminal ganglia, which to our knowledge is the first to be reported. Sixty-seven out of 85 selected protein spots were successfully identified using matrix-assisted laser desorption/ionization mass spectrometry. This reference map was used to monitor changes in the ganglia proteome induced during inflammation in vivo and ex vivo. In vivo we found that FCA treatment specifically induced differential protein expression of two unidentified protein spots and, to a lower extent, of beta-tubulin. Image analysis of ganglia treated ex vivo with the cocktail of cytokines indicated that some of the changes in the protein population were also observed in vivo after FCA treatment. If the cytokine stimulation was performed in the presence of acetaminophen (paracetamol), the drug seemed to reverse the effects of cytokine treatment for at least some protein spots, restoring the same protein pattern observed in control samples.

Animals↗
PubMed2004

UniProt archive.

UniProt Archive (UniParc) is the most comprehensive, non-redundant protein sequence database available. Its protein sequences are retrieved from predominant, publicly accessible resources. All new and updated protein sequences are collected and loaded daily into UniParc for full coverage. To avoid redundancy, each unique sequence is stored only once with a stable protein identifier, which can be used later in UniParc to identify the same protein in all source databases. When proteins are loaded into the database, database cross-references are created to link them to the origins of the sequences. As a result, performing a sequence search against UniParc is equivalent to performing the same search against all databases cross-referenced by UniParc. UniParc contains only protein sequences and database cross-references; all other information must be retrieved from the source databases.

Amino Acid Sequence↗
PubMed1999

Discrimination of the native from misfolded protein models with an energy function including implicit solvation.

An essential requirement for theoretical protein structure prediction is an energy function that can discriminate the native from non-native protein conformations. To date most of the energy functions used for this purpose have been extracted from a statistical analysis of the protein structure database, without explicit reference to the physical interactions responsible for protein stability. The use of the statistical functions has been supported by the widespread belief that they are superior for such discrimination to physics-based energy functions. An effective energy function which combined the CHARMM vacuum potential with a Gaussian model for the solvation free energy is tested for its ability to discriminate the native structure of a protein from misfolded conformations; the results are compared with those obtained with the vacuum CHARMM potential. The test is performed on several sets of misfolded structures prepared by others, including sets of about 650 good decoys for six proteins, as well as on misfolded structures of chymotrypsin inhibitor 2. The vacuum CHARMM potential is successful in most cases when energy minimized conformations are considered, but fails when applied to structures relaxed by molecular dynamics. With the effective energy function the native state is always more stable than grossly misfolded conformations both in energy minimized and molecular dynamics-relaxed structures. The present results suggest that molecular mechanics (physics-based) energy functions, complemented by a simple model for the solvation free energy, should be tested for use in the inverse folding problem, and supports their use in studies of the effective energy surface of proteins in solution. Moreover, the study suggests that the belief in the superiority of statistical functions for these purposes may be ill founded.

Models, Molecular↗
PubMed2001

A two-dimensional map and database of soluble nuclear proteins from HepG2 cells as reference for identification of nutrient-regulated transcription factors.

BACKGROUND: Eukaryotic cells of higher organisms are able to regulate gene transcription in response to changes in the supply of nutrients. In hepatocytes, extracellular glucose levels affect transcription of genes that encode enzymes engaged in glycolysis, gluconeogenesis and lipogenesis. While glucose response elements have been located within a few model gene promoters, the identity of glucose-sensing transcription factors and the mechanisms of their activation remain to be elucidated. AIM OF THE STUDY: We intended to establish a two-dimensional map of nuclear proteins as a reference for identification of nutrient-regulated transcription factors. METHODS: Human hepatoma HepG2 cells were used for the preparation of nuclear extracts. 150-200 microg of the protein mixture were analyzed by 2-dimensional gel electrophoresis (2-DE) and silver-stained protein spots were identified by MALDI-TOF mass spectrometry. RESULTS: Nuclear extracts capable of transcriptional initiation and elongation and containing low amounts of cytoplasmic contaminations were prepared. 543 spots between 17 and 100 kDa and pI 3.7 and 8.8 have been resolved. From these, 65 spots were analyzed by MALDI-TOF mass spectrometry and 53 spots were identified as known proteins of which six represented transcription factors. Regulation by glucose was shown for the activator protein-1 component cJun. Since cJun was not visible on the silver stained 2-DE gel, western blotting of 1-DE gels and immunological detection had to be used in this case. The data were used to construct an online database. CONCLUSIONS: A 2-DE map and database of soluble nuclear proteins is presented. The identification of several transcription factors was possible on the silver-stained gels. However, further fractionation of the nuclear extracts will facilitate the detection of larger numbers of transcriptional regulators. The database and 2-DE map shown here may provide a useful reference for the identification of transcription factors from liver nuclei that are activated by different stimuli, e. g., nutrients.

Carcinoma, Hepatocellular↗