Project description:Glycoproteomics is a powerful yet analytically challenging research tool. Software packages aiding the interpretation of complex glycopeptide tandem mass spectra have appeared, but their relative performance remains untested. Conducted through the HUPO Human Glycoproteomics Initiative, this community study, comprising both developers and users of glycoproteomics software, evaluates solutions for system-wide glycopeptide analysis. The same mass spectrometrybased glycoproteomics datasets from human serum were shared with participants and the relative team performance for N- and O-glycopeptide data analysis was comprehensively established by orthogonal performance tests. Although the results were variable, several high-performance glycoproteomics informatics strategies were identified. Deep analysis of the data revealed key performance-associated search parameters and led to recommendations for improved 'high-coverage' and 'high-accuracy' glycoproteomics search solutions. This study concludes that diverse software packages for comprehensive glycopeptide data analysis exist, points to several high-performance search strategies and specifies key variables that will guide future software developments and assist informatics decision-making in glycoproteomics.
Project description:In pathogenic bacteria, iron acquisition is important for colonization and proliferation in the host under iron-limited conditions. The ability of Vibrio spp. to acquire iron is often critical to their virulence, causing gastroenteritis or excessive watery diarrhea in humans. In the study described here, we cloned the 2,100-bp heme utilization protein gene hupO in Vibrio fluvialis. HupO had high homology to iron-regulated outer membrane receptor proteins in Vibrio sp. and contained motifs that are common to bacterial heme receptors, including a consensus TonB box, a FRAP domain, and an NPNL domain. To characterize the hemin-binding activity of HupO, we purified the recombinant HupO protein (rHupO) from Escherichia coli by using an overexpression system. HupO was found to bind to hemin but not to hemoglobin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting demonstrated that the 77-kDa outer membrane protein HupO of V. fluvialis was induced under iron-restricted conditions. We constructed a hupO mutant, HP1, to investigate the biochemical function of HupO in V. fluvialis. The hemolytic activity of HP1 was reduced compared to that of wild-type cells and, when exposed to hydrogen peroxide, significantly lower numbers of HP1 survived than was the case in the wild type. These results suggest that HupO is associated with virulence expression in V. fluvialis through stimulation of hemolysin production and resistance to oxidative stress. In experimentally infected mice, the 50% lethal dose value of the wild-type was lower than that of the mutant, HP1.
Project description:Neurons isolated from Aplysia californica , an organism with a well-defined neural network, were imaged with secondary ion mass spectrometry, C(60)-SIMS. A major lipid component of the neuronal membrane was identified as 1-hexadecyl-2-octadecenoyl-sn-glycero-3-phosphocholine [PC(16:0e/18:1)] using tandem mass spectrometry (MS/MS). The assignment was made directly off the sample surface using a C(60)-QSTAR instrument, a prototype instrument that combines an ion source with a commercial electrospray ionization/matrix-assisted laser desorption ionization (ESI/MALDI) mass spectrometer. Normal phase liquid chromatography mass spectrometry (NP-LC-MS) was used to confirm the assignment. Cholesterol and vitamin E were also identified with in situ tandem MS analyses that were compared to reference spectra obtained from purified compounds. In order to improve sensitivity on the single-cell level, the tandem MS spectrum of vitamin E reference material was used to extract and compile all the vitamin E related peaks from the cell image. The mass spectrometry images reveal heterogeneous distributions of intact lipid species, PC(16:0e/18:1), vitamin E, and cholesterol on the surface of a single neuron. The ability to detect these molecules and determine their relative distribution on the single-cell level shows that the C(60)-QSTAR is a potential platform for studying important biochemical processes, such as neuron degeneration.
Project description:Characterization of the human blood plasma proteome is critical to the discovery of routinely useful clinical biomarkers. We used an accurate mass and time (AMT) tag strategy with high-resolution mass accuracy cLC-FT-ICR MS to perform a global proteomic analysis of pilot study samples as part of the HUPO Plasma Proteome Project. HUPO reference serum and citrated plasma samples from African Americans, Asian Americans, and Caucasian Americans were analyzed, in addition to a Pacific Northwest National Laboratory reference serum and plasma. The AMT tag strategy allowed us to leverage two previously published "shotgun" proteomics experiments to perform global analyses on these samples in triplicate in less than 4 days total analysis time. A total of 722 (22% with multiple peptide identifications) International Protein Index redundant proteins, or 377 protein families by ProteinProphet, were identified over the six individual HUPO serum and plasma samples. The samples yielded a similar number of identified redundant proteins in the plasma samples (average 446 +/- 23) as found in the serum samples (average 440 +/- 20). These proteins were identified by an average of 956 +/- 35 unique peptides in plasma and 930 +/- 11 unique peptides in serum. In addition to this high-throughput analysis, the AMT tag approach was used with a Z-score normalization to compare relative protein abundances. This analysis highlighted both known differences in serum and citrated plasma such as fibrinogens, and reproducible differences in peptide abundances from proteins such as soluble activin receptor-like kinase 7b and glycoprotein m6b. The AMT tag strategy not only improved our sample throughput but also provided a basis for estimated quantitation.
Project description:[NiFe]-hydrogenases are regulated by various factors to fulfill their physiological functions in bacterial cells. The photosynthetic purple sulfur bacterium Thiocapsa roseopersicina harbors four functional [NiFe]-hydrogenases: HynSL, HupSL, Hox1, and Hox2. Most of these hydrogenases are functionally linked to sulfur metabolism, and thiosulfate has a central role in this organism. The membrane-associated Hup hydrogenases have been shown to play a role in energy conservation through hydrogen recycling. The expression of Hup-type hydrogenases is regulated by H2 in Rhodobacter capsulatus and Cupriavidus necator; however, it has been shown that the corresponding hydrogen-sensing system is nonfunctional in T. roseopersicina and that thiosulfate is a regulating factor of hup expression. Here, we describe the discovery and analysis of mutants of a putative regulator (HupO) of the Hup hydrogenase in T. roseopersicina. HupO appears to mediate the transcriptional repression of Hup enzyme synthesis under low-thiosulfate conditions. We also demonstrate that the presence of the Hox1 hydrogenase strongly influences Hup enzyme synthesis in that hup expression was decreased significantly in the hox1 mutant. This reduction in Hup synthesis could be reversed by mutation of hupO, which resulted in strongly elevated hup expression, as well as Hup protein levels, and concomitant in vivo hydrogen uptake activity in the hox1 mutant. However, this regulatory control was observed only at low thiosulfate concentrations. Additionally, weak hydrogen-dependent hup expression was shown in the hupO mutant strain lacking the Hox1 hydrogenase. HupO-mediated Hup regulation therefore appears to link thiosulfate metabolism and the hydrogenase network in T. roseopersicina.
Project description:Glycoproteome contains valuable information where biomarkers may be discovered for disease diagnosis and monitoring. Nowadays, with the ever-increasing performances of mass spectrometers, the emphasis is shifting to the sample preparation for better throughput and reproducibility. Therefore, to facilitate high throughput N-linked glycopeptide isolation, in this study, a novel hydrazide tip was devised and an integrated workflow of N-linked glycopeptide isolation using hydrazide tips was presented. With the use of bovine fetuin as a standard glycoprotein, the incubation time was determined for each major step of glycopeptide isolation. With the use of commercially available human serum, multiple parallel isolations of glycopeptides were performed using hydrazide tips with a liquid handling robotic system. We demonstrated that, with the hydrazide tips, the processing time was significantly decreased from 3 to 4 days to less than 8 h with excellent reproducibility. The hydrazide pipet tips have great potential in achieving automation of N-linked glycopeptide isolation for high-throughput sample preparation when used in combination with liquid handling robotic systems.
Project description:After an acquired brain injury (ABI), the person remains with several impairments and disabilities that cause a decrease in his/her quality of life (QoL), which could change over time. The objective of the study was to analyse the evolution patterns of QoL in a sample of persons with ABI for one-year as well as the differences in proxy- and self-report versions of a QoL instrument.<h4>Method</h4>The sample comprised 402 persons with ABI with ages ranging between 18 and 91?years, whom 36.20% had had the accident recently (i.e., three years or less). Patients, professionals and relatives responded at three evaluation points to the CAVIDACE scale, an ABI-specific QoL tool.<h4>Results</h4>ANOVAs showed an improvement in QoL in the two follow-ups; the improvement was especially significant in the period between baseline and six months. The respondent factor did not interact with the evaluation time, but significant differences were found between respondents, with scores of patients higher than that for proxies. Finally, the QoL's evolution interacts with the time elapsed since injury, showing significant improvements in the most recent group (i.e., three years or less).<h4>Conclusions</h4>QoL must be considered from the earliest moments after ABI to obtain more significant improvements.
Project description:Glycopeptide antibiotics have long served as drugs of last resort for the treatment of antibiotic-resistant gram-positive bacterial infections. Resistance to the clinically relevant glycopeptides, vancomycin and teicoplanin, threatens to undermine the usefulness of this important class of antibiotics. DNA extracted from a geographically diverse collection of soil samples was screened by PCR for the presence of sequences related to OxyC, an oxidative coupling enzyme found in glycopeptide biosynthetic gene clusters. Every soil sample examined contained at least 1 unique OxyC gene sequence. In an attempt to access the biosynthetic gene clusters associated with these OxyC sequences, a 10,000,000-membered environmental DNA (eDNA) megalibrary was created from a single soil sample. Two unique glycopeptide gene clusters were recovered from this eDNA megalibrary. Using the teicoplanin aglycone and the 3 sulfotransferases found in one of these gene clusters, mono-, di-, and trisulfated glycopeptide congeners were produced. The high frequency with which OxyC genes were found in environmental samples indicates that soil eDNA libraries are likely to be a rewarding source of glycopeptide gene clusters. Enzymes found in these gene clusters should be useful for generating new glycopeptides analogs. Environmental DNA megalibraries, like the one constructed for this study, can provide access to many of the natural product biosynthetic gene clusters that are predicted to be present in soil microbiomes.