Journal of Integrated -OMICS
Not a member yet
215 research outputs found
Sort by
Proteome response to heat stress in the Antarctic clam Laternula elliptica: DOI: 10.5584/jiomics.v3i1.125
No full textThe proteome can be regarded as a molecular phenotype, as changes in protein expression patterns have a direct effect on organismal physiology and fitness. The analysis of the proteome can therefore be an invaluable tool for our understanding of the mechanisms underlying phenotypic changes in response to environmental change. However, proteomic studies on thermal stress in marine species have mainly focused on heat shock protein expression, and little information is available for other components of the cellular stress response. This is particularly limiting for Antarctic species, which can lack the ability to induce heat shock protein expression in response to experimentally induced heat stress. The present study analysed changes in protein expression patterns in the Antarctic clam Laternula elliptica after exposure to elevated temperatures using two dimensional gel electrophoresis and mass spectrometry. Acute exposure to elevated temperatures had an effect in global protein expression patterns, suggesting that L. elliptica has the capacity to alter protein expression in response to heat stress. Changes in the expression of 14 proteins out of 264 analysed were observed in response to different levels of heat stress. Four of the 14 proteins had database matches and were identified as the cytoskeletal protein tubulin and associated chaperone TCP-1, and the enzymes enolase and aldehyde dehydrogenase, part of the minimal stress proteome and involved in redox regulation. 
Red Blood Cell Lipidomics analysis through HPLC-ESI-qTOF: application to red blood cell storage: DOI: 10.5584/jiomics.v3i1.123
No full textRecent developments in mass spectrometry (MS) have enabled fast and sensitive detection of lipid species in different biological matrices.
In the present study we performed an on-line HPLC-microTOF-Q MS approach to the red blood cell (RBC) lipidome. We thus exploited bioinformatic tools for the interrogation of novel databases, such as LIPID MAPS. By means of ad hoc software suites for mass spectrometry-based metabolomics analyses, we could address the key biological issue of the RBC lipidome, within the framework of RBC storage for transfusion purposes. Samples were collected from subjects living in the province of Viterbo, where olive oil consumption represents a central aspect of the diet. On this ground, we could postulate a diet specific effect on the accumulation of lipid-specific storage lesions.
The analyses yielded the tentative identification of a huge number of lipid molecules on the basis of accurate intact mass values and retention times, and MS/MS validation. This analytical workflow was exploited to consolidate existing knowledge on the RBC lipid composition and individuate statistically significant fluctuations of lipids throughout storage duration of RBC concentrates under blood bank conditions. Our analysis indicated ceramides, glycerophospholipids and sterols as key targets of RBC storage lesions to the lipidome, that will deserve further targeted investigations in the future. It also emerged how compositional analyses of the RBC lipidome might end up yielding different results on the basis of the background of the blood donor (i.e. diet), which might translate into region-specific lipidomic alterations over storage progression of RBC concentrates
-Omics fields of study related to plant-parasitic nematodes: DOI: 10.5584/jiomics.v3i1.120
No full textPlant-parasitic nematodes (PPN) cause significant losses and these pathogens must be addressed amid the growing demand for food, global warming, and the discarded use of inorganic pesticides. For these reasons, acquiring deeper knowledge about PPN and devising new management strategies are important in order to meet future food demand. This review focuses on PPN and their applicable and diverse –omics fields of study. While most efforts have been centered on transcriptomics, other –omics studies have recently begun to expand. The few genomes sequenced (Meloidogyne incognita, M. hapla, and Bursaphelenchus xylophilus) have shown high diversity in PPN. This review also discusses the future prospects and uses of –omics relative to PPN
Analysis of the rat primary hepatocyte nuclear proteome through sub-cellular fractionation: DOI: 10.5584/jiomics.v2i2.102
No full text
Characterising primary hepatocytes and their de-differentiation in culture is vital for the refinement of current culture techniques and for the development of new and improved in vitro hepatocyte models. We have performed multiplexed iTRAQ proteomics on whole cell preparations and further employed nuclear fractionation to expand the coverage of this important organelle. We identify many proteins that change in abundance during culture of rat hepatocytes for 48h and map their molecular functions. 431 proteins were identified and quantified in whole cell homogenates, mapping to 69 molecular functions using the PANTHER (Protein ANalysis THrough Evolutionary Relationships) classification system. In whole cell homogenates liver-associated functions, such as oxidoreductase activity, were enriched compared with the reference rat proteome dataset but some functions, such as transcriptional activity, were under-represented. Nuclear fractionation resulted in the identification of an additional 156 proteins which mapped to 31 molecular functions. These proteins included some associated with hepatic differentiation, such as HNF4alpha and CCAAT/enhancer-binding protein beta and others with less well-defined roles.
Hierarchical clustering of samples within each experiment showed segregation of fresh and cultured sample types and stringent statistical analysis demonstrated significant changes in 36% of proteins from the whole cell homogenates and 21% of proteins from the nuclear dataset (adjusted p value
Elucidation of carbon transfer in a mixed culture of Acidiphilium cryptum and Acidithiobacillus ferrooxidans using protein-based stable isotope probing: DOI: 10.5584/jiomics.v2i1.85
No full textAlthough many examples of syntrophic cultures are known, details of carbon utilization and carbon transfers within them often remain elusive due to limitations in methods used to detect carbon flux. We have applied the recently developed method of protein-based stable isotope probing (protein-SIP) to track carbon flow in a mixed culture of acidophilic bacteria. The heterotroph Acidiphilium cryptum was grown in the presence of 13C-labeled galactose, together with the iron- and sulfur-oxidizing autotroph Acidithiobacillus ferrooxidans. Cultures were harvested at five time points, proteins extracted and separated by 1-dimensional SDS gel electrophoresis, peptides obtained by tryptic digest of gel slices and analyzed by UPLC Orbitrap MS/MS measurements. Syntrophic interactions were confirmed by analysis of the time-dependent incorporation of 13C into peptides, and quantified by calculation of relative isotope abundance (RIA) and labeling ratio (lr) from mass spectral isotope patterns. 13CO2 formed by catabolism of galactose by A. cryptum, was found to be assimilated by At. ferrooxidans which used tetrathionate as electron donor. Mass spectral data indicated that 13C-labeled organic substances, mostly peptides, secreted by the chemoautotroph were assimilated by the heterotroph. The data provided unequivocal evidence for two-way transfer of carbon in mixed cultures of autotrophic and heterotrophic acidophilic bacteria
Proteomic analysis of bronchoalveolar lavage fluid in an equine model of asthma during a natural antigen exposure trial: DOI: 10.5584/jiomics.v2i2.112
No full textBackground
Heaves is a complex, asthma-like respiratory disease that affects many older horses. While environmental and genetic components to the disease have been proposed, the specific pathophysiology of heaves is still poorly understood. Using proteomic techniques, we compared the protein profile of bronchoalveolar lavage fluid (BALF) in the lungs of healthy horses and horses affected with heaves.
Methods
Clinical signs of the disease were induced in heaves-affected horses using an experimental hay exposure model. Samples of BALF were collected from all horses before and after the hay exposure trial. Mass spectrometry (LC-MS) was used to evaluate the differences in the global BALF peptide profile between the control and heaves-affected horses. Tandem mass spectrometry (LC-MS/MS) was used to identify differentially expressed proteins in the two groups of horses. The identification of two proteins was validated with Western blot assays.
Results
One hundred peptides were differentially expressed between healthy controls and heaves-affected horses; 76 peptides were over-expressed in controls and 24 were over-expressed in heaves-affected horses. The identifications of transferrin and secretoglobin were confirmed with Western blot.
Conclusions
This study demonstrates that proteomics can be used to compare the protein profiles of BALF from healthy and diseased horses. These techniques may prove helpful in determining the pathophysiology of complex disease
Identifying Core Protein Complexes from Downscaled Tandem Affinity Purifications: DOI: 10.5584/jiomics.v2i1.81
No full textIn this study we show that via stable retroviral-expression of tagged EGFR in the PC9 lung cancer cell line and stable doxycycline-inducible expression of tagged Grb2 using a flp-mediated recombination HEK293 cell system, the SH-TAP can be downscaled to 5 to 12.5 mg total protein input (equivalent to 0.5 - 1 × 15 cm culture plate or 4 - 8 × 106 cells). The major constituents of the EGFR complex (USB3B, GRB2, ERRFI, HSP7C, GRP78, HSP71) and the Grb2 complex (ARHG5, SOS1, ARG35, CBL, CBLB, PTPRA, SOS2, DYN2, WIPF2, IRS4) were identified. Adjustment of the quantity of digested protein injected into the mass spectrometer reveals that optimisation is required as high quantities of material led to a decrease in protein sequence coverage and the loss of some interacting proteins. This investigation should aid other researchers in performing tandem affinity purifications in general, and in particular, from low quantities of input material. From this study, we believe that it is now possible to map protein-protein interaction networks from cells that are difficult to cultivate in large quantities (i.e., primary cells) or cells from which it is not feasible to generate a stable cell line without using viral expression systems or the flp-FRT recombination system. Such studies would be of considerable benefit in delineating disease-related signalling pathways in aberrant tissues obtained from mouse models and/or human patient material
Secretome differences between the taxonomically related but clinically differing mycobacterial species Mycobacterium abscessus and M. chelonae: DOI: 10.5584/jiomics.v2i2.98
No full textRapidly growing non-tuberculous mycobacteria (NTM) are significant human pathogens which show high inter-species differences in clinical characteristics (virulence, host immune response) during infection even within a given NTM complex. Understanding the differences between the secreted proteomes of the member species for an NTM complex may reveal the basis of their differential virulence and host pathogenesis potential including host immune reactions. In this study, major secreted proteins of the two taxonomically close but clinically differing member species M. abscessus and M. chelonae of the M. chelonae-M. abscessus(MCA) complex were compared using an approach based on 2-dimensional gel electrophoresis (2-DE) and MALDI-TOF analyses. The two secretomes showed dramatic differences. Of the 73 major secreted proteins identified, majority were expressed in a species-specific manner, including 37 in M. chelonae and 32 in M. abscessus. Interestingly, 9 of these differentially expressed proteins were orphan proteins showing homology to either hypothetical proteins or those with no defined function. The other 60 distinctly expressed proteins were homologs of those associated with various bacterial cellular functions and virulence, namely cell wall synthesis or lipid metabolism, metabolic and respiratory pathways, stress response and signal transduction, gene regulation, and immune response. This information on species-specific secreted proteins would help understand the critical virulence factors and host pathogenesis mechanisms in these mycobacterial species and provide the basis for developing better therapeutic strategies. These proteins may also serve as potential targets for species-specific diagnosis as an additional outcome. To our knowledge, this is the first attempt to characterize the secretome of M. chelonae (for which the genome sequence is not yet available) and the secretome differences between M. abscessus and M. chelona
In silico directed mutagenesis using software for glycosylation sites prediction as a new step in antigen design: DOI: 10.5584/jiomics.v2i1.80
No full textIn silico directed mutagenesis with the aim to estimate consequences of mutational pressure on number of N- and O-glycosylation sites has been proposed as an important step in antigen design. Using this kind of methodology one is able to estimate probabilities at which N- and O-glycosylation sites can be destroyed and created due to one-step missense mutations in the subsequent gene. Mutational AT-pressure has been simulated in the region of env gene coding for HIV1 gp120. Consequences of 741 amino acid substitutions have been predicted with the help of NetNGlyc 1.0 and NetOGlyc 3.1 software. The probability of O-glycosylation site destruction (2.16%) in HIV1 gp120 protein due to a single missense GC to AT mutation in env gene is higher than the probability of a new site creation (0.40%). The probability of N-glycosylation site destruction in HIV1 gp120 protein is equal to the probability of its creation (5.53%), while the number of N-glycosylation sites which can be created due to a single missense GC to AT mutation in env gene is 1.27 times higher than the number of N-glycosylation sites which can be destroyed
An Improved Isotope-coded Affinity Tag Technology for Thiol Redox Proteomics: DOI: 10.5584/jiomics.v2i1.83
No full textIsotope coded affinity tag (ICAT) is a gel-free technology for quantitative proteomics. In ICAT procedure, strong cation exchange chromatography (SCX) using increased potassium chloride gradient is recommended for peptide fractionation. Here we report optimization of hydrophilic interaction chromatography (HILIC) as an alternative strategy for peptide fractionation of ICAT samples. HILIC exhibits high separation efficiency and does not require any downstream desalting steps. Compared to SCX based ICAT, integration of HILIC into the ICAT technology has resulted in high rates of protein identification, cysteine mapping, and quantification of cysteine-containing peptides. The improved technology has shown utility in thiol redox proteomics. Interestingly, results from HILIC ICAT and SCX ICAT are complementary. Implementation of both provides high coverage analysis of a complex proteome