Identifier mapping performance for integrating transcriptomics and proteomics experimental results.

BACKGROUND: Studies integrating transcriptomic data with proteomic data can illuminate the proteome more clearly than either separately. Integromic studies can deepen understanding of the dynamic complex regulatory relationship between the transcriptome and the proteome. Integrating these data dictates a reliable mapping between the identifier nomenclature resultant from the two high-throughput platforms. However, this kind of analysis is well known to be hampered by lack of standardization of identifier nomenclature among proteins, genes, and microarray probe sets. Therefore data integration may also play a role in critiquing the fallible gene identifications that both platforms emit. RESULTS: We compared three freely available internet-based identifier mapping resources for mapping UniProt accessions (ACCs) to Affymetrix probesets identifications (IDs): DAVID, EnVision, and NetAffx. Liquid chromatography-tandem mass spectrometry analyses of 91 endometrial cancer and 7 noncancer samples generated 11,879 distinct ACCs. For each ACC, we compared the retrieval sets of probeset IDs from each mapping resource. We confirmed a high level of discrepancy among the mapping resources. On the same samples, mRNA expression was available. Therefore, to evaluate the quality of each ACC-to-probeset match, we calculated proteome-transcriptome correlations, and compared the resources presuming that better mapping of identifiers should generate a higher proportion of mapped pairs with strong inter-platform correlations. A mixture model for the correlations fitted well and supported regression analysis, providing a window into the performance of the mapping resources. The resources have added and dropped matches over two years, but their overall performance has not changed. CONCLUSIONS: The methods presented here serve to achieve concrete context-specific insight, to support well-informed decisions in choosing an ID mapping strategy for "omic" data merging.

Proteomic analysis of stage I endometrial cancer tissue: identification of proteins associated with oxidative processes and inflammation.

OBJECTIVE: The present study aimed to identify differentially expressed proteins employing a high resolution mass spectrometry (MS)-based proteomic analysis of endometrial cancer cells harvested using laser microdissection. METHODS: A differential MS-based proteomic analysis was conducted from discrete epithelial cell populations gathered by laser microdissection from 91 pathologically reviewed stage I endometrial cancer tissue samples (79 endometrioid and 12 serous) and 10 samples of normal endometrium from postmenopausal women. Hierarchical cluster analysis of protein abundance levels derived from a spectral count analysis revealed a number of proteins whose expression levels were common as well as unique to both histologic types. An independent set of endometrial cancer specimens from 394 patients were used to externally validate the differential expression of select proteins. RESULTS: 209 differentially expressed proteins were identified in a comparison of stage I endometrial cancers and normal post-menopausal endometrium controls (Q<0.005). A number of differentially abundant proteins in stage I endometrial cancer were identified and independently validated by western blot and tissue microarray analyses. Multiple proteins identified with elevated abundance in stage I endometrial cancer are functionally associated with inflammation (annexins) and oxidative processes (peroxiredoxins). PRDX1 and ANXA2 were both confirmed as being overexpressed in stage I cancer compared to normal endometrium by independent TMA (Q=0.008 and Q=0.00002 respectively). CONCLUSIONS: These data provide the basis for further investigation of previously unrecognized novel pathways involved in early stage endometrial carcinogenesis and provide possible targets for prevention strategies that are inclusive of both endometrioid and serous histologic subtypes.

Synovial fluid proteins differentiate between the subtypes of juvenile idiopathic arthritis.

OBJECTIVE: Juvenile idiopathic arthritis (JIA) is a heterogeneous group of inflammatory diseases, and no clinically useful prognostic markers to predict disease outcome in children with JIA are currently available. Synovial fluid likely reflects the proteins present in the inflamed synovium. The purpose of this study was to delineate the synovial fluid proteome and determine whether protein expression differs in the different subtypes of JIA. METHODS: Synovial fluid samples obtained from children with oligoarticular JIA, polyarticular JIA, or systemic JIA were compared. Two-dimensional gel electrophoresis for protein separation and matrix-assisted laser desorption ionization-time-of-flight mass spectrometry and quadripole time-of-flight mass spectrometry for protein identification were used for this study. Synovial fluid cells were analyzed by polymerase chain reaction (PCR) for the presence of haptoglobin messenger RNA (mRNA). RESULTS: The synovial fluid proteome of the samples was delineated. The majority of proteins showed overexpression in JIA synovial fluid as compared with noninflammatory control samples. There were 24 statistically significantly differentially expressed spots (>2-fold change; P < 0.05) between the subtypes of JIA. PCR analysis revealed haptoglobin mRNA, suggesting that haptoglobin is locally produced in an inflamed joint in JIA. CONCLUSION: Despite the similar histologic appearance of inflamed joints in patients with different subtypes of JIA, there are differences in protein expression according to the subtype of JIA. Haptoglobin is differentially expressed between the subtypes of JIA and is locally produced in an inflamed joint in JIA. Haptoglobin and other differentially expressed proteins may be potential biomarkers in JIA.

All-or-none N-glycosylation in primate follicle-stimulating hormone beta-subunits.

Human FSH exists as two major glycoforms designated, tetra-glycosylated and di-glycosylated hFSH. The former possesses both alpha- and beta-subunit carbohydrates while the latter possesses only alpha-subunit carbohydrate. Western blotting differentiated the glycosylated, 24,000 M(r) hFSHbeta band from the non-glycosylated 21,000 M(r) FSHbeta band. Postmenopausal urinary hFSH preparations possessed 75-95% 24,000 M(r) hFSHbeta, while pituitary hFSH immunopurified from 21- to 43-year-old females and 21-43-year-old males possessed only 35-40% 24,000 M(r) hFSHbeta. The pituitary hFSH from a postmenopausal woman on estrogen replacement was 75% 21,000 M(r) hFSHbeta. Other immunopurified postmenopausal pituitary hFSH preparations possessed 50-60% 21,000 M(r) hFSHbeta. Gel filtration removed predominantly 21,000 M(r) free hFSHbeta and reduced its abundance to 13-22% in postmenopausal pituitary hFSH heterodimer preparations. A major regulatory mechanism for FSH glycosylation involves control of beta-subunit N-glycosylation, possibly by inhibition of oligosaccharyl transferase. Two primate species exhibited the same all-or-none pattern of pituitary FSHbeta glycosylation.

Site-specific glycosylation of an aglycosylated human IgG1-Fc antibody protein generates neoglycoproteins with enhanced function.

A range of well-defined IgG glycoforms was prepared by employing a combination of synthetic carbohydrate chemistry and genetic engineering. The key aspect of this methodology is the coupling of thioaldoses with cysteine-containing proteins to give disulfide-linked neoglycoproteins. This technology was applied to the synthesis of a series of synthetic N-glycan thioaldoses which were coupled to an aglycosylated IgG1-Fc fragment, engineered to have Cys-297 in place of glycan-linked Asn (Deltah-Fc N297C). Analysis of the resulting Fc neoglycoproteins by mass spectrometry and trypsin digestion showed that the saccharides were site-selectively incorporated at Cys-297 to full occupancy without affecting other Fc protein disulfides. The neoglycoproteins were tested for their ability to interact with human FcgammaRI by inhibiting superoxide production by gamma-interferon-stimulated U937 cells. The neoglycoproteins displayed enhanced superoxide inhibition relative to aglycosylated Deltah-Fc N297C, where increased glycan size correlated positively with increased inhibition.

Phase variable changes in genes lgtA and lgtC within the lgtABCDE operon of Neisseria gonorrhoeae can modulate gonococcal susceptibility to normal human serum.

The alpha-chain of the core oligosaccharide of the lipo-oligosaccharide (LOS) produced by Neisseria gonorrhoeae can undergo reversible and rapid changes in structure due to phase-variable production of certain enzymes employed in the biosynthesis of the lacto-N-neotetraose structure. Five of these enzymes are encoded by the lgtABCDE operon, and polynucleotide tracts within three of these genes (lgtA, lgtC and lgtD) can be substrates for slipped-strand mispairing events that lead to nucleotide insertions or deletion events which result in variable production of their respective gene products. We now report that phase-variable synthesis of the lgtA and lgtC gene products in strain FA19 results in the production of elongated LOS alpha-chains and that the presence of these LOS species can result in gonococci being sensitive to the bacteriolytic action of serum-antibody and complement. Hence, phase variation within the lgtABCDE operon can significantly impact the ability of gonococci to subvert this important host defense system.

Characterization and estimation of proanthocyanidins and other phenolics in coffee pulp (Coffea arabica) by thiolysis-high-performance liquid chromatography.

Fresh and 3-day-old coffee pulp of the Arabica variety were analyzed for polyphenol composition followed by characterization by two different methods. The first method consisted in subjecting coffee pulp powder to direct thiolysis. For the second method, coffee pulp was subjected to successive solvent extractions, followed by thiolysis. Quantification of phenolic compounds was then achieved by high-performance liquid chromatography (HPLC) analysis of thiolysis products. Four major classes of polyphenols were identified: flavan-3-ols (monomers and procyanidins), hydroxycinnamic acids, flavonols, and anthocyanidins. Differences in concentration of procyanidins were observed between fresh and 3-day-old coffee pulp. Constitutive units were mainly epicatechin, representing more than 90% of the proanthocyanidin units, with average degrees of polymerization in the range of 3.8-9.1. Monomer to hexamer units of flavan-3-ols from fresh coffee pulp were separated by normal-phase HPLC. Molecular size of oligomeric proanthocyanidins was obtained by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Results obtained confirm the presence of oligomers of the flavan-3-ol (-)-epicatechin.

Initial proteome analysis of caffeine-induced proteins in Aspergillus tamarii 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.

Characterization of a novel lipid-A from Rhizobium species Sin-1. A unique lipid-A structure that is devoid of phosphate and has a glycosyl backbone consisting of glucosamine and 2-aminogluconic acid.

The structure of the lipid-A from Rhizobium species Sin-1, a nitrogen-fixing Gram-negative bacterial symbiont of Sesbania, was determined by composition, nuclear magnetic resonance spectroscopic, and mass spectrometric analyses. The lipid-A preparation consisted of a mixture of structures due to differences in fatty acylation and in the glycosyl backbone. There were two different disaccharide backbones. One disaccharide consisted of a distal glucosaminosyl residue beta-linked to position 6 of a proximal 2-aminoglucono-1,5-lactonosyl residue, and in the second disaccharide, the proximal residue was 2-amino-2,3-dideoxy-d-erythro-hex-2-enono-1,5-lactone. For both disaccharides, the distal glucosamine was acylated at C-2' primarily with beta-hydroxypalmitate (beta-OHC16:0) which, in turn, was O-acylated with 27-hydroxyoctacosanoic acid. For some of the lipid-A molecules, the distal glucosaminosyl residue was also acylated at C-3' with beta-hydroxymyristate (beta-OHC14:0), whereas other molecules were devoid of this acyl substituent. Both the 2-aminoglucono-1,5-lactonosyl and 2-amino-2,3-dideoxy-d-erythro-hex-2-enono-1,5-lactonosyl residues were acylated at C-2, primarily with beta-OHC16:0. Minor amounts of lipid-A molecules contained beta-OHC14:0 at C-3 and/or beta-hydroxystearate (beta-OHC18:0) or beta-hydroxyoctadecenoate (beta-OHC18:1) as the C-2 and C-2' N-acyl substituents.

The membrane phospholipids of Neisseria meningitidis and Neisseria gonorrhoeae as characterized by fast atom bombardment mass spectrometry.

The phospholipids of Neisseria meningitidis and Neisseria gonorrhoeae were characterized by fast atom bombardment (FAB)-MS and GLC-MS. The major phospholipids were phosphatidylethanolamine (PE), followed by phosphatidylglycerol (PG), with minor amounts of phosphatidic acid (PA) and trace levels of cardiolipin (DPG). All of the phospholipid preparations were variable in their fatty acyl substituents, which included C16:1, C16:0, C18:1, C14:0, C14:1 and C12:0. By MS/MS analysis, all pathogenic Neisseria spp. phospholipids contained a saturated fatty acyl substituent and either a saturated or unsaturated fatty acyl substituent in the sn-1 and sn-2 positions, respectively. Compared with enteric bacterial species, the phospholipids of N. meningitidis and N. gonorrhoeae have increased levels of phospholipids with short-chain fatty acyl residues (i.e. increases in C12:0, C14:1 and C14:0) and variable amounts of C18:1. The percentage of total PE and PG molecules with the shorter-chain fatty acids ranges from 35 to 47% and 42 to 66%, respectively, for N. meningitidis while these respective values are <10% and <5% for Escherichia coli. The variability and variety of meningococcal and gonococcal phospholipids suggest novel genetic mechanisms of neisserial phospholipid assembly and regulation, which may be important for the biology and pathogenesis of N. meningitidis and N. gonorrhoeae.

Characterization of a tomato protein that inhibits a xyloglucan-specific endoglucanase.

A basic, 51 kDa protein was purified from suspension-cultured tomato and shown to inhibit the hydrolytic activity of a xyloglucan-specific endoglucanase (XEG) from the fungus Aspergillus aculeatus. The tomato (Lycopersicon esculentum) protein, termed XEG inhibitor protein (XEGIP), inhibits XEG activity by forming a 1 : 1 protein:protein complex with a Ki approximately 0.5 nm. To our knowledge, XEGIP is the first reported proteinaceous inhibitor of any endo-beta-1,4-glucanase, including the cellulases. The cDNA encoding XEGIP was cloned and sequenced. Database analysis revealed homology with carrot extracellular dermal glycoprotein (EDGP), which has a putative role in plant defense. XEGIP also has sequence similarity to ESTs from a broad range of plant species, suggesting that XEGIP-like genes are widely distributed in the plant kingdom. Although Southern analysis detected only a single XEGIP gene in tomato, at least five other XEGIP-like tomato sequences have been identified. Similar small families of XEGIP-like sequences are present in other plants, including Arabidopsis. XEGIP also has some sequence similarity to two previously characterized proteins, basic globulin 7S protein from soybean and conglutin gamma from lupin. Several amino acids in the XEGIP sequence, notably 8 of the 12 cysteines, are generally conserved in all the XEGIP-like proteins we have encountered, suggesting a fundamental structural similarity. Northern analysis revealed that XEGIP is widely expressed in tomato vegetative tissues and is present in expanding and maturing fruit, but is downregulated during ripening.

KpsF is the arabinose-5-phosphate isomerase required for 3-deoxy-D-manno-octulosonic acid biosynthesis and for both lipooligosaccharide assembly and capsular polysaccharide expression in Neisseria meningitidis.

We have identified and defined the function of kpsF of Neisseria meningitidis and the homologues of kpsF in encapsulated K1 and K5 Escherichia coli. KpsF was shown to be the arabinose-5-phosphate isomerase, an enzyme not previously identified in prokaryotes, that mediates the interconversion of ribulose 5-phosphate and arabinose 5-phosphate. KpsF is required for 3-deoxy-d-manno-octulosonic acid (Kdo) biosynthesis in N. meningitidis. Mutation of kpsF or the gene encoding the CMP-Kdo synthetase (kpsU/kdsB) in N. meningitidis resulted in expression of a lipooligosaccharide (LOS) structure that contained only lipid A and reduced capsule expression in the five invasive disease-associated meningococcal serogroups (A, B, C, Y, and W-135). The step linking meningococcal capsule and LOS biosynthesis was shown to be Kdo production as the expression of capsule was wild type in a Kdo transferase (kdtA) mutant. Thus, in addition to lipooligosaccharide assembly, Kdo is required for meningococcal capsular polysaccharide expression. Furthermore, N. meningitidis, unlike enteric Gram-negative bacteria, can survive and synthesize only unglycosylated lipid A.