A common open representation of mass spectrometry data and its application to proteomics research

A broad range of mass spectrometers are used in mass spectrometry (MS)-based proteomics research. Each type of instrument possesses a unique design, data system and performance specifications, resulting in strengths and weaknesses for different types of experiments. Unfortunately, the native binary data formats produced by each type of mass spectrometer also differ and are usually proprietary. The diverse, nontransparent nature of the data structure complicates the integration of new instruments into preexisting infrastructure, impedes the analysis, exchange, comparison and publication of results from different experiments and laboratories, and prevents the bioinformatics community from accessing data sets required for software development. Here, we introduce the 'mzXML' format, an open, generic XML (extensible markup language) representation of MS data. We have also developed an accompanying suite of supporting programs. We expect that this format will facilitate data management, interpretation and dissemination in proteomics research.     

Cloning and characterization of an mRNA encoding a novel G protein alpha-subunit abundant in mantle and gill of pearl oyster Pinctada fucata

Nacre formation is an ideal model to study biomineralization processes. Although much has been done about biomineralization mechanism of nacre, little is known as to how cellular signaling regulates this process. We are interested in whether G protein signaling plays a role in mineralization. Degenerate primers against conserved amino acid regions of G proteins were employed to amplify cDNA from the pearl oyster Pinctada fucata. As a result, the cDNA encoding a novel G(s)alpha (pfG(s)alpha) from the pearl oyster was isolated. The G(s)alpha cDNA encodes a polypeptide of 377 amino acid residues, which shares high similarity to the octopus (Octopus vulgaris) G(s)alpha. The well-conserved A, C, G (switch I), switch II functional domains and the carboxyl terminus that is a critical site for interaction with receptors are completely identical to those from other mollusks. However, pfG(s)alpha has a unique amino acid sequence, which encodes switch III and interaction sites of adenylyl cyclase respectively. In situ hybridization and Northern blotting analysis revealed that the oyster G(s)alpha mRNA is widely expressed in a variety of tissues, with highest levels in the outer fold of mantle and epithelia of gill, the regions essential for biomineralization. We also show that overexpression of the pfG(s)alpha in mammalian MC3T3-E1 cells resulted in increased cAMP levels. Mutant pfG(s)alpha that has impaired CTX substrate diminished its ability to induce cAMP production. Furthermore, the alkaline phosphatase (ALP) activity, an indicator for mineralization, is induced by the G(s)alpha in MC3T3-E1 cells. These results indicated that G(s)alpha may be involved in regulation of physiological function, particularly in biological biomineralization.     

Using immobilized metal affinity chromatography, two-dimensional electrophoresis and mass spectrometry to identify hepatocellular proteins with copper-binding ability

To further our knowledge of intracellular copper transport, we used a proteomics strategy to search for hepatic proteins with copper-binding ability. Hep G2 cytosolic and microsomal fractions were applied to a copper(II)-loaded immobilized metal-affinity chromatography (IMAC) column. Protein identification was performed with 2-D gel electrophoresis and mass spectrometry. We identified 48 cytosolic proteins and 19 microsomal proteins displaying copper-binding ability. These proteins are diverse in function. Fifty-two of the 67 proteins contain putative metal-binding domains. We have identified many components of the Hep G2 copper metalloproteome including a large number of proteins not previously known to bind copper.     

TRAIL-R2 (DR5) mediates apoptosis of synovial fibroblasts in rheumatoid arthritis

TRAIL has been proposed as an anti-inflammatory cytokine in animal models of rheumatoid arthritis (RA). Using two agonistic mAbs specific for TRAIL-R1 (DR4) and TRAIL-R2 (DR5), we examined the expression and function of these death receptors in RA synovial fibroblast cells. The synovial tissues and primary synovial fibroblast cells isolated from patients with RA, but not those isolated from patients with osteoarthritis, selectively expressed high levels of cell surface DR5 and were highly susceptible to anti-DR5 Ab (TRA-8)-mediated apoptosis. In contrast, RA synoviocytes did not show increased expression of TRAIL-R1 (DR4), nor was there any difference in expression of Fas between RA and osteoarthritis synovial cells. In vitro TRA-8 induced apoptosis of RA synovial cells and inhibited production of matrix metalloproteinases induced by pro-inflammatory cytokines. In vivo TRA-8 effectively inhibited hypercellularity of a SV40-transformed RA synovial cell line and completely prevented bone erosion and cartilage destruction induced by these cells. These results indicate that increased DR5 expression and susceptibility to DR5-mediated apoptosis are characteristic of the proliferating synovial cells in RA. As highly proliferative transformed-appearing RA synovial cells play a crucial role in bone erosion and cartilage destruction in RA, the specific targeting of DR5 on RA synovial cells with an agonistic anti-DR5 Ab may be a potential therapy for RA.     

Inhibition of an activated Ras protein with genetically selected peptide aptamers

Mutant alleles of Ras maintain an activated, GTP-bound conformation and relay mitogenic signals that cannot be turned off. A genetic selection in Saccharomyces cerevisiae was used to identify peptide aptamers that suppress the growth arrest phenotype of an activated Ras allele. Peptide aptamers were expressed as C-terminal fusions to glutathione-S-transferase. Modifications that alter the coding capacity of the peptide aptamer indicate it is necessary for Ras2-Val19 suppression. Aptamer expression also reduces the elevated levels of cAMP and suppresses the heat shock sensitivity characteristic of Ras-activated yeast cells. The peptide aptamer retains suppressor activity when fused to thioredoxin. The peptide aptamer expression strategy described here indicates that aptamers presented as unconstrained peptides have functional capacity in vivo.     

Genus-specific protein binding to the large clusters of DNA repeats (short regularly spaced repeats) present in Sulfolobus genomes

Short regularly spaced repeats (SRSRs) occur in multiple large clusters in archaeal chromosomes and as smaller clusters in some archaeal conjugative plasmids and bacterial chromosomes. The sequence, size, and spacing of the repeats are generally constant within a cluster but vary between clusters. For the crenarchaeon Sulfolobus solfataricus P2, the repeats in the genome fall mainly into two closely related sequence families that are arranged in seven clusters containing a total of 441 repeats which constitute ca. 1% of the genome. The Sulfolobus conjugative plasmid pNOB8 contains a small cluster of six repeats that are identical in sequence to one of the repeat variants in the S. solfataricus chromosome. Repeats from the pNOB8 cluster were amplified and tested for protein binding with cell extracts from S. solfataricus. A 17.5-kDa SRSR-binding protein was purified from the cell extracts and sequenced. The protein is N terminally modified and corresponds to SSO454, an open reading frame of previously unassigned function. It binds specifically to DNA fragments carrying double and single repeat sequences, binding on one side of the repeat structure, and producing an opening of the opposite side of the DNA structure. It also recognizes both main families of repeat sequences in S. solfataricus. The recombinant protein, expressed in Escherichia coli, showed the same binding properties to the SRSR repeat as the native one. The SSO454 protein exhibits a tripartite internal repeat structure which yields a good sequence match with a helix-turn-helix DNA-binding motif. Although this putative motif is shared by other archaeal proteins, orthologs of SSO454 were only detected in species within the Sulfolobus genus and in the closely related Acidianus genus. We infer that the genus-specific protein induces an opening of the structure at the center of each DNA repeat and thereby produces a binding site for another protein, possibly a more conserved one, in a process that may be essential for higher-order stucturing of the SRSR clusters.     

Phenotypic features of Ferroplasma acidiphilum strains Yt and Y-2

Earlier, we described a new family of mesophilic, strictly autotrophic Fe(2+)-oxidizing archaebacteria, Ferroplasmaceae, which belongs to the order Thermoplasmales and includes the genus Ferroplasma and species F. acidiphilum (strain YT) [1]. The present work is concerned with a comparative study of phenotypic characteristics of the type strain YT and a new strain, F. acidiphilum Y-2, isolated from dense pulps produced during oxidation of arsenogold concentrates from the Bakyrchikskoe (Kazakhstan) and Olimpiadinskoe (Krasnoyarsk Krai) ore deposits, respectively. The G + C content of DNA from strains YT and Y-2 comprised 35.1 and 35.2 mol%, respectively; the level of DNA-DNA homology between the strains was 84%. Restriction profiles of chromosomal DNA from both strains exhibited a similarity coefficient of 0.87. Genotypic characteristics of these strains indicate their affiliation to the same species. The cells of both strains are polymorphic and lack cell walls. Strains of F. acidiphilum oxidized ferrous oxide and pyrite as the sole source of energy and fixed carbon dioxide as the sole carbon source. Strains required yeast extract as a growth factor. Optimum pH for cell growth ranged from 1.7 to 1.8; the temperature optima for the growth of strains YT and Y-2 were 34-36 and 40-42 degrees C, respectively. Comparative analysis of total lipids revealed their close similarity in the strains; two glycophospholipids comprised 90% of total lipids: lipid I, beta-D-glucopyranosylcaldarchaetidylglycerol (about 55%), and lipid II, trihexosylcaldarchaetidylglycerol (26%), whose isopranyl chains contained no cyclopentane rings. The carbohydrate fraction of lipid I hydrolysate contained only D-glucose, whereas hydrolysate of lipid II contained both D-glucose and D-galactose in a molar ratio of 2:1. Thus, it was established that the intraspecific phylogenetic divergence within F. acidiphilum is manifested in two the strains by different temperature optima against the background of similarity in other phenotypic properties.