Journal of Integrated -OMICS
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A robust permutation test for quantitative SILAC proteomics experiments: DOI: 10.5584/jiomics.v2i2.109
No full textStable Isotope Labeling by Amino Acids in Cell Culture (SILAC) along with other relative quantitation methods in proteomics have become important tools in the analysis of cellular and subcelluar functions. Although numerous experimental applications of SILAC have been developed, there is no consensus on the use of statistical procedures to analyze the resulting experimental data. SILAC experiments output relative abundance ratios for proteins to quantify dierences in cell populations. These ratios have traditionally been analyzed with fold-change methods and hypothesis testing procedures under Gaussian distribution assumptions.
We find that the normality assumption is invalid and can lead to inaccurate quantitation of the significance of differences between cell populations. As a solution, a permutation based hypothesis test as an alternative for assessing significance is introduced. We develop distribution-free permutation testing methods for assessing SILAC experiments. These tests generate p-values which can be easily interpreted and if necessary, thefalse discovery rate of these p-values can be easily controlled. To compare the permutation test against competing methodology, we used a set of simulations based upon a theoretical model of SILAC ratio data.
Through the simulation studies, we find that the permutation test is generally superior to the competing hypothesis tests across the range of simulation scenarios. We also find that the permutation test is typicallymore powerful and accurate than the competing methods at the five percent level of signicance and averaged over the spectrum of signicance levels. Because of the broad superiority of the permutation test and the easeof implementation, we propose the use of the permutation test as a standard measure of protein signicance in SILAC experiments
A survey on coronary heart disease related signal pathways, drug targets and pharmacological interventions: DOI: 10.5584/jiomics.v2i2.105
No full textCoronary heart disease (CHD), the most common form of cardiovascular disease, is a chronic, multifactorial disease. With significant advances in our understanding of the pathophysiological process of CHD in recent years, more and more drug targets have been identified and adopted in drug discovery for CHD. Purpose and methods: In this review, a comprehensive perspective of pathological and pharmacological development of CHD was introduced through searching in multiple bibliographic sources. Results: CHD related signal pathways (including 412 proteins), drug targets (including 101 proteins) and pharmacological interventions were summarized and visualized. Conclusions: The knowledge of these signal pathways and drug targets may facilitate new drug discovery and medicine intervention for CHD in the future
New insights in Trypanosoma cruzi proteomic map: further post-translational modifications and potential drug targets in Y strain epimastigotes: DOI: 10.5584/jiomics.v2i2.107
No full textChagas´ disease is a neglected sickness endemic in Latin America, caused by the protozoa Trypanosoma cruzi. The current treatment for the disease is unsatisfactory, and the development of potent compounds for novel molecular targets is critical. In this framework, proteomics could be a powerful tool in the evaluation of possible candidates for drug intervention. In this work, a two-dimensional electrophoresis (2-DE) and mass spectrometry (MS) approach was employed in T. cruzi epimastigotes (Y strain). Different gel staining protocols (Coomassie Blue, Pro-Q-Diamond and Pro-Q-Emerald) were performed to assess the protein content and possible post-translational modifications of this parasite. Here, 78 most intense spots were identified by Coomassie staining, 22 by Pro-Q-Diamond (phosphoproteins) and 15 by Pro-Q-Emerald (glycoproteins). Compared with the results of other large-scale T. cruzi proteomic studies, 15 novel proteins were identified here using MALDI-TOF/TOF, and 12 of these have not yet been described at the protein level. The predominant localisation and function of the identified proteins was cytosol and protein metabolism, respectively. Regarding the potentially interesting drug targets, 8 proteins presented no similarity to human sequences and promising characteristics for chemotherapy intervention. Our data provides novel insights in the metabolic pathways of T. cruzi, which could aid in the discovery of alternative drugs for Chagas´ disease
Comparative immunoproteome analysis of the response of susceptible A.BY/SnJ and resistant C57BL/6 mice to Coxsackievirus B3-infection: DOI: 10.5584/jiomics.v2i2.96
No full textBoth, innate and cell-mediated immunity contribute to prevention of chronic myocarditis and consecutively, cardiomyopathy. Thus, in resistant C57BL/6 mice myocarditis induced by Coxsackievirus B3 (CVB3)-infection is abrogated by immune-mediated mechanisms. However, susceptible A.BY/SnJ mice develop dilated cardiomyopathy (DCM) due to chronic myocarditis. Cardiac auto-antibodies have been shown to play a pivotal role in the initiation and/or progression of inflammatory DCM. In order to investigate differences in the autoimmune response of susceptible and resistant mice to infection with CVB3, the patterns of autoantibodies reacting with heart proteins in A.BY/SnJ and C57BL/6 mice were profiled by 2-D Western blot analysis during the acute and chronic phases of myocarditis up to three months, when the pathophysiological phenotype in the susceptible mice has progressed to DCM. In the early phase of infection both mouse strains displayed similar autoantibody patterns. In contrast, at later time points compared to the resistant C57BL/6 strain susceptible A.BY/SnJ mice displayed a much stronger autoimmune response against proteins associated with cell structure, protein transport as well as primary metabolic processes such as energy production. During chronic myocarditis strong antibody responses against myosin heavy chain 6, mitochondrial and heat shock proteins were observed in A.BY/SnJ mice. Antibodies directed against alpha-enolase, serotransferrin, radixin and two processed myosin protein species accumulated late and only in A.BY/SnJ mice suffering from inflammatory DCM. Functional assignment of the target proteins of cardiac autoantibodies indicates that these might be directly involved in cardiac dysfunction
Normalization of protein at different stages in SILAC subcellular proteomics affects functional analysis: DOI: 10.5584/jiomics.v2i2.108
No full textQuantitative subcellular proteomics is a powerful method to interrogate spatial dynamics of cells or tissues. Stable isotope labeling by amino acids in cell culture (SILAC) is a popular quantitative approach that is ideally suited to subcellular proteomics because samples can be combined very early to reduce technical variability in the subcellular fractionation and downstream processing. However, validation of results using orthogonal methods such as immunoblotting do not allow mixing of samples prior to fractionation, leading to potentially different outcomes. Here we have investigated the impact protein normalization before or after subcellular fractionation has on the functional analysis and experimental conclusions. As a model system, we compared the detergent-resistant membrane (DRM) fraction of mouse embryonic fibroblasts (MEF) from caveolin-1-null mice with wildtype controls. Caveolin-1 is cholesterol-binding protein which is essential for formation of plasma membrane caveolae, a subtype of lipid raft membrane microdomains. Surprisingly, we found that the relative protein content of DRM as a percentage of total protein content is 1.6 fold higher for Cav1-/- MEF compared to wild type MEF, leading to different SILAC ratios in pre fractionation mix and post fractionation mix experiments. Most of the observed differences were replicated by mathematical modeling of the normalization effect, with the striking exception for mitochondrial DRM proteins. Interestingly, caveolin-1 affected DRM proteins in the post fractionation mix data showed a significant enrichment of the mitochondrial oxidative phosphorylation pathway, which was not observed in the pre fractionation mix experiment. The observed quantitative changes in mitochondrial DRM proteins using different analyses suggest a caveolin-1 induced change rather than simple contamination, and may support recent reports of caveolin-1-dependent mitochondrial cholesterol changes. Based on these results, we recommend a thorough understanding of how experimental conditions impact relative subcellular fraction in order to make an informed decision on the most appropriate point to combine SILAC samples for quantitative subcellular proteomic analysis
Comparative proteomic map among vanA-containing Enterococcus isolated from yellow-legged gulls: DOI: 10.5584/jiomics.v2i1.86
No full text
The increase of VRE, therefore, represents an urgent threat for patient care and creates a reservoir of mobile resistance genes for other, more virulent pathogens. The existence of VRE in different ecologic niches complicates the understanding of its epidemiology. The aim of the present study was to study the proteome of 2 vanA strains recovered from seagull faecal samples. The vanA E. durans and vanA E. faecium isolates presented different genomic patterns: tet(M)-tet(L)-erm(B) and tet(M)-tet(L)-erm(B)-hyl, respectively. A total of 123 spots were excised from two-dimensional gel electrophoresis (2-DE) gel of vanA E. durans SG 2 strain, and 16 were successfully identified by using MS, representing 42 different proteins. For the vanA E. faecium SG 50 strain, 93 spots were excised from the 2-DE gel and 23 were identified, representing 47 different proteins. The vancomycin/teicoplanin A-type resistance protein vanA in vanA E. durans SG 2 strain was present in two different spots. The identified proteins have shown diverse functional activities including glycolysis, conjugation, translation, protein biosynthesis, among others. This work reports the impact of proteomics on knowledge of vanA enterococci strains and will be helpful to further understand the antibiotic-resistant mechanism
Identification of outer membrane proteins of Edwardsiella tarda in response to high concentration of copper: DOI: 10.5584/jiomics.v2i2.91
No full textThe antimicrobial properties of copper have been reported, but little is known about the outer membrane proteins regulating copper resistance. In the present study, a sub-proteomic approach was utilized to investigate altered OM proteins of Edwardsiella tarda in response to CuSO4. Upregulation of HemR and dwonregulation of Imp, EvpB, TolC, ETAE_2935, ETAE_1480, and EIB_1723 were detected in E. tarda EIB202 survived in 1.0 mM CuSO4 compared with the control without the ion. These alterations were validated, at random, using Western blotting. They were first revealed here to be bacterial copper-resistant proteins in combination of protein homology analysis. These findings highlight the way to clarify copper-resistant mechanisms. 
Molecular Modeling of Cathepsin B protein in different Leishmania strains: DOI: 10.5584/jiomics.v1i1.32
No full textCathepsin B like cysteine proteases representing a major component of the lysosomal proteolytic repertoire plays an important role in intracellular protein degradation. Comparative models of cathepsin B (CatB) protein of six different Leishmania strains were developed using MODELLER. The modeled three-dimensional (3-D) structure has the correct stereochemistry as gauged from the Ramachandran plot and good 3-D structure compatibility as assessed by PROCHECK and the DOPE score (DS2.1, Accelrys). The modeled proteins were energy minimized and validated using standard dynamic cascade protocol (DS 2.1). Seven different disulfide bonding sites are predicted in CatB protein of Leishmania. Two domains were identified and different motifs are present in catB protein of Leishmania like aspargine glycosylation site, protein kinase phosphorylation site, Protein kinase C activation site, N-myristoylation site. Considering that cathepsin B is essential for survival of Leishmania, including for virulence to the mammalian host, it may be viewed as an attractive drug target.  
Proteomic study in an Escherichia coli strain from seagulls of the Berlengas Natural Reserve of Portugal: DOI: 10.5584/jiomics.v1i1.19
No full textThe increasing bacterial resistance among common pathogens is threatening theeffectiveness of several antibiotics. This represents a serious public health problem as such bacterial strains have already been detected in domestic, wild-life animals and humans. Using Escherichia coli as a model organism, we applied a proteomic approach to the topic of antimicrobial resistance. In order to identify and characterize the proteome of extended-spectrum β-lactamase (ESBL) type TEM-52 producing-Escherichia coli strain of a faecal sample taken from Yellow-legged seagulls (Larus cachinnans) a bidimensional electrophoresis (2-DE) technique with an isoelectric focusing followed by a SDS-PAGE, was used. Eighty seven individualized protein spots were identified. All were suitable for peptide mass fingerprinting by a mass spectrometric technique (MALDI/TOF MS). Their identification was carried out by searching appropriate bioinformatic databases. All proteins were related to E. colistrains. Detection of proteins related to several E. coli strains linked with virulent and enterohaemorrhagic consequences in ESBL producing-E. coli isolates of seagull samples raises the question of how such similarities arise bearing in mind these remarkably different microbial ecosystems
A new method for measuring functional similarity of microRNAs: DOI: 10.5584/jiomics.v1i1.21
No full textMicroRNAs (miRNAs) are a group of small RNAs with regulatory roles at post-transcriptional level. Although they have been clustered based on their sequence or structure similarities, there is still no effective method to determine their functional similarities due to the lack of miRNAs functional annotation. To address this critical need, we presented here a novel method for systematic study of functional similarities among human miRNAs by using their target genes GO semantic similarities. The functional similarities were validated by comparing with miRNA expression similarities. To extract the highly significant clusters, we used multi-scale bootstrap re-sampling in clustering miRNAs functional similarities. The clustering of human miRNAs based on target genes molecular function annotation led to 44 significant clusters. The clustering results were coherent with biological knowledge. Our analysis suggests that systematic clustering based on target genes GO semantic similarities can aid to reveal the functional diversity of miRNA families. Additionally, this method can be extended to other species and used to predict novel miRNA functions