Journal of Integrated -OMICS
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Proteomic analysis of low quantities of cellular material in the range obtainable from scarce patient samples: DOI: 10.5584/jiomics.v5i1.172
No full textThe application of proteomics to patient material is increasingly widespread, however, a major shortcoming still are the number of cells or protein material that can be obtained. This study explores the lower limit of cell numbers that can be successfully analysed by liquid chromatography mass spectrometry to determine the protein expression profile that is specific to, and indicative of, the investigated cell type. The aim was to analyse an equivalent quantity of cellular material that can be obtained from, e.g., a fine-needle aspiration biopsy (FNAB). Fifteen thousand and 30,000 cells from adherent (HEK293) and suspension (U937) cell lines were lysed under two different conditions: a ‘native’ and a denaturing buffer. To extend the study to clinical material, human whole PBMCs were also lysed under identical conditions. Proteins from 5,000 and 10,000 cells were analysed by both 1D and 2D-LC-MSMS on an LTQ Orbitrap XL mass spectrometer. In total, 3,219; 1,693 and 659 unique proteins were identified from HEK293, U937 and total PBMCs, respectively. Additionally, an iTRAQ 4-plex experiment was performed to determine the relative quantity of the proteins in the three cell types. In this study, we show that it is feasible to obtain a deep, yet cell-specific protein profile from a very low number of cultured and primary cells. This advancement will enable proteomic-profiling of cellular material from fine needle aspiration biopsies that ultimately can assist cytopathologists in the diagnosis of disease
2D DIGE proteomic analysis of multidrug resistant and susceptible clinical Mycobacterium tuberculosis isolates: DOI: 10.5584/jiomics.v4i1.168
No full textTuberculosis (TB) is the leading cause of infectious disease related mortality worldwide. Infection of Mycobacterium tuberculosis (Mtb) leads to nearly 3 million deaths every year due to tuberculosis. Rifampicin and Isoniazid (RH) are the key drugs to being used for the treatment of tuberculosis. Reports in recent years indicate that the increasing emergence of resistance to these drugs. The resistance to these drugs severely affects options for effective treatment. The current vaccine for tuberculosis has variable protective efficacy and there is no commercially available serodiagnostic test for this disease with acceptable sensitivity and specificity for routine laboratory use, especially in case of multidrug resistance. In order to develop a new diagnostic tool for detection of Mtb, multidrug resistant Mtb as well and improve the tuberculosis vaccine, it is necessary to indentify novel antigenic candidates, especially in identification of multidrug resistant associated protein antigens. Here, we present a 2-D gel-based proteomic survey of the changes in RH resistant Mtb. The proteins extracted from RH resistant and susceptible Mtb clinical isolates were analyzed by two-dimensional differential in gel electrophoresis (2D-DIGE). Protein intensities of 41 spots were found to be regulated in RH resistant isolates. A total of 28 proteins were identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Twelve proteins of interest are NADH-dependent enoyl-[acyl-carrier-protein] reductase, 60 kDa chaperonin 2, Chaperone protein DnaK, 3-oxoacyl-(Acyl-carrier-protein0 reductase, Probable acetyl-CoA acyltransferase FadA2, two Acetyl/propionyl-CoA carboxylase, alpha subunit, Universal stress protein Rv1636/MT1672, Dihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydrogenase complex, Glutamine synthetase 1 and two uncharacterized proteins (Rv2557 and Rv1505c)
Subtoxic concentrations of benzo[a]pyrene induce metabolic changes and oxidative stress in non-activated and affect the mTOR pathway in activated Jurkat T cells: DOI: 10.5584/jiomics.v4i1.157
No full textAlthough the activation of immune cells is the first and thereby pivotal step in the immunological cascade, the current knowledge about the details of this process is quite limited. Recent studies have shown that aromatic compounds, such as B[a]P, influence the immune system even at low concentrations. We investigated the effect of a subtoxic B[a]P concentration (50 nM) on the proteome and the metabolome of non-activated and activated Jurkat T cells. The GeLC-MS/MS analysis yielded 2624 unambiguously identified proteins. In addition to typical regulatory pathways associated with T cell activation, pathway analysis by Ingenuity IPA revealed several metabolic processes, for instance purine and pyruvate metabolism. The activation process seems to be influenced by B[a]P suggesting an important role of the mTOR pathway in the cellular adaptation. B[a]P exposure of non-activated Jurkat cells induced signaling pathways such as protein ubiquitination and NRF2 mediated oxidative stress response as well as metabolic adaptation involving pyruvate, purine and fatty acid metabolism. Thus, we validated the proteome results by determining the concentrations of 183 metabolites with FIA-MS/MS and IC-MS/MS. Furthermore, we were able to show that Jurkat cells metabolize B[a]P to B[a]P-1,6-dione. The combined evaluation of proteome and metabolome data with an integrated, genome-scale metabolic model provided novel systems biological insights into the complex relation between metabolic and proteomic processes in Jurkat T cells during activation and subtoxic chemical exposure
Molecular cloning and protein characterization of a heme-binding globin predicted in a sugar cane EST database: DOI: 10.5584/jiomics.v4i1.156
No full textA very large and representative sugar cane expression sequence tag (EST) library (SUCEST) was sequenced by a Brazilian consortium, opening the possibility to study important proteins, such as hemoglobins, which are largely present across the plant kingdom. The widespread presence and long evolutionary history of plant hemoglobins suggest a major role for this protein family in plants; however, little is known about their functional roles. In this study, we report the identification and characterization of a putative non-symbiotic hemoglobin cDNA clone that was identified in SUCEST. The cDNA was cloned, and the recombinant protein was purified and folded, as shown by its circular dichroism and emission fluorescence spectra. The expressed globin protein was able to bind hemin, as a characteristic Soret band was observed in the absorbance spectrum and increases were seen in the amount of secondary structure and in the stability of the protein. A model for the structure of the sugarcane hemoglobin was created using the crystal structure of a rice hemoglobin, and this model showed a conserved globular conformation
An integrated proteomic and physiological approach to understand the adhesion mechanism of the probiotic Lactobacillus reuteri Lb26 DSM16341: DOI: 10.5584/jiomics.v3i2.143
No full textThe adhesion ability of the probiotic Lactobacillus reuteri Lb26 DSM16341 was tested to both enterocyte-like Caco-2 cells and to extracellular matrix proteins (laminin, fibronectin and collagen I and IV). The adhesiveness was lost after an alkaline treatment known to release moonlighting proteins from lactobacillar cell surface. To characterize the putative adhesive molecules, a 2-DE experiment in the pI range 4-7 was performed on the extracellular proteins. The expression of several moonlighting proteins involved in adhesion (i.e. GAPDH, EF-Tu, phosphoglycerate kinase) was demonstrated. Some of the identified adhesins were able to bind plasminogen (Plg), but did not convert it into plasmin (Plm), in absence of exogenous activators. This indicates that the moonlighting proteome of L. reuteri Lb26 DSM16341 can contribute to adhesion processes
Comparison of preservation methods for bacterial cells in cytomics and proteomics: DOI: 10.5584/jiomics.v3i1.115
No full textCell sampling during long-term experiments usually requires reliable storage of cells for later analysis. In this study, we evaluated three different fixation or preservation strategies (sodium azide fixation, deep freezing and vacuum drying) with regard to their effects on bacterial cells. Pseudomonas putida was used as a model organism and stored for shorter (2 d) and longer periods (28 d). The impact of the treatments (preservation method, duration) was evaluated on the level of single cells using flow cytometry and on the population level using protein mass spectrometry. The effect of sodium azide fixation on scatter parameters and DNA content of bacterial cells was small (1.01 ≤ sd ≤ 1.76) for short term and larger for long term storage (1.59 ≤ sd ≤ 2.33), as determined by FlowFP fingerprinting. In contrast, deep frozen and vacuum dried samples showed properties highly similar to fresh reference samples, even after extended storage (0.5 ≤ sd ≤ 1.2). The mass spectrometric analysis revealed about 800 proteins for each storage condition and the proteome profiles evaluated by label-free quantification showed characteristic differences. The variation of protein quantity within functional groups was lowest for deep frozen and vacuum dried cell samples (sd log2 relative quantity < 1) and only marginally increased after 28 d. In conclusion, deep freezing was found to be the method of choice, but simple vacuum drying of cells with storage at 4°C can be a convenient alternative
Proteomic changes in extended-spectrum beta-lactamase-producing Escherichia coli strain under cefotaxime selection: DOI: 10.5584/jiomics.v3i2.140
No full textProteomics can be used to study the metabolic pathways and mechanisms involved in antimicrobial resistance. The aim of this comparative proteomic study was to establish the overall changes in the proteome of a naturally occurring ESBL-producing E. coli strain (C5478) stressed with its minimal inhibitory concentration (2 μg/mL) of cefotaxime, compared to the proteome of the same strain without antimicrobial stress, by using 2-D gel electrophoresis (2-DE) followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The comparative proteomic analysis revealed that the abundance of numerous protein species changed in the strain stressed by CTX compared to the non-stressed wild-type strain. A total of 188 spots were excised from the 2-DE gel of the wild-type strain, 112 of which were successfully identified by MALDI-TOF MS, representing 110 different proteins. Concerning the 2-DE gel of the CTX-stressed bacteria, 171 spots were excised and 156 were identified, representing 143 different proteins. The proteins identified in both strains were categorized according to their biological functions. These proteins were involved in metabolism, protein synthesis, cell division, stress responses, and antimicrobial resistance, among others. These findings will be helpful to further understand not only the antimicrobial resistance mechanisms, but also the role of wild animals as reservoirs in spreading antimicrobial-resistant bacteria into the environment
Global proteomic profiling of the membrane compartment of bovine testis cell populations: DOI: 10.5584/jiomics.v3i2.136
No full text
Spermatogonial stem cells hold enormous potential in mammalian reproductive medicine through the preservation of gametes, the restoration of fertility, enhancement of germ-lineage genetic manipulation and the improvement in our understanding of stem cell biology. Here we describe the protein profiles of the membrane compartment of bovine testicular cell isolates which were enriched for germ cells using differential plating. The isolated cells were characterised with antibodies to UCHL1 (previously known as PGP9.5) for type A spermatogonia; DDX4 (previously known as VASA) for germ cells and vimentin for Sertoli cells. Ultracentrifugation techniques were used to specifically isolate cell membranes, with membrane protein identifications significantly increased when compared to whole cell lysates. We utilised the filter-aided sample preparation protocol for improved digestion efficiency of membrane proteins. Using ESI-LC-MS/MS, we compared the proteins present in two cell populations. A total of 1,387 proteins were identified in bovine testis cell isolates, of which 39% were membrane-associated. A total of 64 proteins were differentially expressed in the non-adhered fraction (enriched for undifferentiated germ cells), of which 16 were unique to this cell population and the remaining 48 showed a two-fold change as judged by spectral counting. This analysis revealed a number of candidate germ cell markers including the known markers, DDX4 and UCHL1. The proteomic profiles generated in this study support and complement transcriptomics studies and reinforce the potential of proteomics in identifying and characterising the protein effectors of self-renewal and/or differentiation in stem cells
Cellular Protein/Peptide Expression Profiles (PEPs): an alternative approach for easy identification of cyanobacterial species: DOI: 10.5584/jiomics.v3i2.138
No full text
Cyanobacterial harmful algal blooms (CyanoHABs) are recognized as an expanding and serious global problem that threatens human health. Timely and accurate identification of cyanobacteria is of vital importance for public health. Morphologic characteristics of cyanobacteria have been used for classical taxonomic studies and identification purposes. However, misidentification may occur either due to subjective judgment by the operators or inability to recognize natural variations of morphotypes. To circumvent problems of morphology-based identification methods, we reported previously a rapid and simple method for the identification of dinoflagellates using protein/peptide expression profiles (PEPs) of whole cell protein extracts generated by MALDI-TOF-MS (Lee FWF et. al., 2008). In the present study, we applied this method in the identification of harmful cyanobacteria. Our results showed that various species of the cyanobacteria can be easily distinguished from each other using their PEPs
Determining the C-Terminal Amino Acid of a Peptide from MS/MS Data: DOI: 10.5584/jiomics.v3i2.137
No full textProteomics is currently chiefly based on mass spectrometry (MS) which is the tool of choice to investigate proteins. Two computational approaches to derive the tandem mass spectrum precursor’s sequence are widely employed. Database search essentially retrieves the sequence by matching the spectrum to all entries in a database whereas de novo sequencing does not depend on a sequence database. Both approaches benefit from knowledge about the enzyme used to generate the peptides. Most algorithms default to trypsin for its abundant usage. Trypsin cuts after arginine and lysine and thus the c-terminal amino acid is not known precisely and usually either of the two. Furthermore, 90% of protein terminal peptides may not end with either arginine or lysine and may thus contain any of the other amino acids. Here an algorithm is presented which predicts the c-terminal amino acid to be arginine, lysine or any other.
Here an algorithm, named RKDecider, to sort the c-terminal amino acid into one of three groups (arginine, lysine, and other) is presented. Although around 90% accuracy was achieved during data mining spectra for rules that determine the c-terminal amino acid, the implementation’s (RKDecider) accuracy is a little less and achieves about 80%. This is due to the fact that the decision trees were implemented as a rule-based system for speed considerations. The implementation is freely available at: http://bioinformatics.iyte.edu.tr/RKDecider