Current Proteomics - Volume 16, Issue 4, 2019

Neuroproteomics, as a sub-discipline of proteomics, has enlightened the pathway for the study of different complicated diseases and brain disorders. Since four decades, various analytical and quantitative techniques have been used to cure problems related to brain and memory. Brain has a complex structure with various cells and cell types, the expressing proteins and suppressing factors too. Drug addiction is one of the main health concerns as it causes physiological changes in brain and affects its different parts. Some of these drugs like cocaine, marijuana, nicotine and alcohol not only affect memory and brain cells but also lead to expression and suppression of unwanted and beneficial proteins respectively. A variety of techniques involving separation techniques, quantification techniques and analytical techniques are used along with the combination of bioinformatics and magical tools for analyzing different aspects of brain parts especially proteome of the brain cells. Moreover, different animal models preferably those resembling human beings are routinely used in neuroproteomics to study the effects of different drugs on the brain proteome. Different experiments have already been performed by the researchers on drug abuse that helped massively in estimating not only the effects of drug addiction on the brain of highly complex organisms (human beings) but also to propose different therapeutics.

Background: Mass spectrometry is a tool used in analytical chemistry to identify components in a chemical compound and it is of tremendous importance in the field of biology for high throughput analysis of biomolecules, among which protein is of great interest. Objective: Advancement in proteomics based on mass spectrometry has led the way to quantify multiple protein complexes, and proteins interactions with DNA/RNA or other chemical compounds which is a breakthrough in the field of bioinformatics. Methods: Many new technologies have been introduced in electrospray ionization (ESI) and Matrixassisted Laser Desorption/Ionization (MALDI) techniques which have enhanced sensitivity, resolution and many other key features for the characterization of proteins. Results: The advent of ambient mass spectrometry and its different versions like Desorption Electrospray Ionization (DESI), DART and ELDI has brought a huge revolution in proteomics research. Different imaging techniques are also introduced in MS to map proteins and other significant biomolecules. These drastic developments have paved the way to analyze large proteins of >200kDa easily. Conclusion: Here, we discuss the recent advancement in mass spectrometry, which is of great importance and it could lead us to further deep analysis of the molecules from different perspectives and further advancement in these techniques will enable us to find better ways for prediction of molecules and their behavioral properties.

Background: Plasminogen is a blood plasma glycoprotein of molecular weight about 92,000 Daltons. Physiologically, it incorporates into blood clots and after its activation by plasminogen activators to plasmin can perform a fibrinolytic function. Microplasmin is truncate polypeptide chain derivate of plasmin may be increase the fibrinolytic activity. Objective: To study the amino acid sequence of two polypeptides chains derivate to the plasminogen with fibrinolytic activity. Methods: The two polypeptides chains were prepared by isoelectric precipitation of human plasma in sodium borate buffer. The sample in a second step was subjected to affinity and ionic interchange chromatography and denaturalized electrophoresis was carried out on the sample previous heat 70ºC. Results: Two polypeptide chains of 29.000 and 35.000 Daltons by autolysis controlled were obtained with 25 UI of fibrinolytic activity in fibrin plate. Conclusion: Microplasmin was obtained with cleavage in different amino acid bounds and rearrangement of amino acids by autolysis with controlled alkaline precipitation.

Background: Cerebrospinal fluid (CSF) contains pro-growth factors that can affect proliferation, migration and differentiation of Mesenchymal Stem Cells (MSCs). Objective: This study aimed to isolate MSC like cells from CSF of patients with meningioma and psudotumorcerebri (PTC) and identify differentially expressed proteins in these cells. Methods: Five patients with newly diagnosed intracranial meningioma and five patients with PTC were recruited in this comparative proteomics study. MSCs were isolated from CSF and validated by mesenchyml and non-mesenchyml fluorochrome antibodies, and flow cytometer analysis. Two- Dimensional Gel Electrophoresis (2-DE) coupled with Mass Spectrometry (MS) was performed to identify differentially expressed proteins. Results: Microscopic views of the isolated cells as well as flow cytometer analysis were found to be compatible with MSC-like cells. Eight distinct protein spots were differentially and reproducibly expressed among the stained gels of two studied groups. The identified proteins were Phosphoglycerate Mutase 1 (PGAM1), LIM and SH3 domain protein (LASP1), peroxiredoxin-6 (PRDX-6), type I cytoskeletal 9 (KRT9), Superoxide Dismutase (SOD), endoplasmin, Stathmin 1 (STMN1), and glutathione S-transferase (GST). Conclusion: This study provides new insights into the plausible role of CSF derived MSCs in cancer progression, and reveals a promising therapeutic opportunity for targeting of MSC proteins in patients with meningioma.

Background: Bioethanol derived from lignocellulosic biomass can supplement the ethanol supplies in a sustainable manner. However, the bioethanol production process is still not cost effective and researchers are looking for novel strategies like simultaneous saccharification fermentation to cut down the production cost. Thermotolerant yeast Saccharomyces cerevisiae JRC6 is reported to improve the fermentation efficiency under SSF. However, the mechanism of thermotolerance of the strain is unknown which is important for developing more robust yeast strains for simultaneous saccharification and fermentation. Objective: To identify proteomic changes responsible for imparting thermotolerance by iTRAQ based profiling of Saccharomyces cerevisiae JRC6 by growing at optimum (30°C) and high temperature (40°C). Methods: iTRAQ labeling followed by electrospray ionization based tandem mass spectrometry using SCIEX 5600 Triple-TOF Mass Spectrometer (MS). Results: A total of 582 proteins involved in heat shock, metabolism, biosynthesis, transport of biomolecules, cell division, etc. were identified. Cells grown at 40°C showed many-fold increase in the expression for many proteins involved in different functions specially biosynthesis, heat stress and metabolism. At 40°C heat shock proteins (78), prefoldin subunit (6), DNA binding protein SNT1, J type co-chaperone JAC1, elongation factor 1-β, glutathione synthase, malate synthase (2), purine biosynthesis protein ADE17, SSD1 protein, alcohol dehydrogenase 1, 3, 60S ribosomal protein L35-B, mitochondrial import protein MAS5 and many other proteins were significantly upregulated. Conclusion: The iTRAQ analysis revealed many heat shock proteins and heat stable alcohol dehydrogenases which can be exploited to develop a more robust yeast strain suitable for simultaneous saccharification and fermentation or consolidated bioprocessing.

Background: The molecular mechanisms or valuable biomarkers for early diagnosis of colorectal cancer (CRC) are not fully elucidated yet. Objective: To understand the proteomic changes at the global level in the carcinogenesis of CRC, differentially expressed proteins between normal intestinal epithelial cells CCD841 and colorectal cancer cells HCT116 were identified. Method: The isobaric tags for relative and absolute quantitation (iTRAQ) coupled with 2D LC-MS/MS proteomic approach were performed for screening the altered proteins between cells CCD841 and HCT116. Results: A total of 1947 proteins were identified after filtering and using a 1% false discovery rate. Based on a final cutoff (> 3.16 and < 0.32), 229 proteins were found to be significantly altered, among which 95 (41%) were up-regulated while 134 (59%) were down-regulated. Gene Ontology analysis revealed that the differentially expressed proteins were mainly cell part proteins involved in cellular process and binding in terms of subcellular distribution, biological process, and molecular function. KEGG analysis indicated that the differentially expressed proteins were significantly involved in the process of focal adhesion, pathogenic Escherichia coli infection, leukocyte transendothelial migration, bacterial invasion of epithelial cells, regulation of actin cytoskeleton, DNA replication and so on. Conclusion: Collectively, our data identified differentially expressed proteins in colon cancer carcinogenesis, which could provide the clues on unraveling the molecular mechanism of CRC.

Background: DAB389IL-2 (Denileukin diftitox) as an immunotoxin is a targeted pharmaceutical protein and is the first immunotoxin approved by FDA. It is used for the treatment of various kinds of cancer such as CTCL lymphoma, melanoma, and Leukemia but among all of these, treatment of CTCL has special importance. DAB389IL-2 consists of two distinct parts; the catalytic domain of Diphtheria Toxin (DT) that genetically fused to the whole IL-2. Deamidation is the most important reaction for chemical instability of proteins occurs during manufacture and storage. Deamidation of asparagine residues occurs at a higher rate than glutamine residues. The structure of proteins, temperature and pH are the most important factors that influence the rate of deamidation. Methods: Since there is not any information about deamidation of DAB389IL-2, we studied in silico deamidation by Molecular Dynamic (MD) simulations using GROMACS software. The 3D model of fusion protein DAB389IL-2 was used as a template for deamidation. Then, the stability of deamidated and native form of the drug was calculated. Results: The results of MD simulations were showed that the deamidated form of DAB389IL-2 is more unstable than the normal form. Also, deamidation was carried by incubating DAB389IL-2, 0.3 mg/ml in ammonium hydrogen carbonate for 24 h at 37o C in order to in vitro experiment. Conclusion: The results of in vitro experiment were confirmed outcomes of in silico study. In silico and in vitro experiments were demonstrated that DAB389IL-2 is unstable in deamidated form.

Background: Yaks inhabit high-altitude are well-adapted to the hypoxic environments. Though, the mechanisms involved in regulatory myocardial protein expression at high-altitude were not completely understood. Objective: To revel the molecular mechanism of hypoxic adaptation in yak, here we have applied comparative myocardial proteomics in between yak and cattle by isobaric Tag for Relative and Absolute Quantitation (iTRAQ) labelling. Methods: To understand the systematic protein expression variations in myocardial tissues that explain the hypoxic adaptation in yak, we have performed iTRAQ analysis combined with Liquid Chromatography- Tandem Mass Spectrometry (LC-MS/MS). Bioinformatics analysis was performed to find the association of these Differentially Expressed Proteins (DEPs) in different functions and pathways. Protein to protein interaction was analyzed by using STRING database. Results: 686 Differentially Expressed Proteins (DEPs) were identified in yak with respect to cattle. From which, 480 DEPs were up-regulated and 206 were down-regulated in yak. Upregulated expression of ASB4, STAT, HRG, RHO and TSP4 in yak may be associated with angiogenesis, cardiovascular development, response to pressure overload to heart and regulation of myocardial contraction in response to increased oxygen tension. The up-regulation of mitochondrial proteins, ACAD8, GPDH-M, PTPMT1, and ALDH2, may have contributed to oxidation within mitochondria, hypoxia-induced cell metabolism and protection of heart against cardiac ischemic injuries. Further, the upregulated expression of SAA1, PTX, HP and MBL2 involved in immune response potentially helpful in myocardial protection against ischemic injuries, extracellular matrix remodeling and free heme neutralization/ clearance in oxygen-deficient environment. Conclusion: Therefore, the identification of these myocardial proteins in will be conducive to investigation of the molecular mechanisms involved in hypoxic adaptations of yaks at high-altitude condition.

Background: In eukaryotic cells, apoptosis signaling pathways are controlled mainly by aspartic acid cysteine proteases (caspases). However, certain unicellular microorganisms, such as Giardia duodenalis, lack these proteins. Thus, other cysteine proteases may play an important role in the parasite apoptosis signaling pathway. Objective: To understand the effect of cathepsin B-like inhibition on the cell viability of Giardia duodenalis and its cell death process. Methods: Bioinformatics analysis was performed to identify apoptotic proteases. Analysis showed that cathepsin B-like protease genes from G. duodenalis were the best candidate. A homology modeling technique was used to explore in silico the inhibitory effect of E-64 against cathepsin B-like proteases from G. duodenalis genome and to examine the effect of curcumin on cathepsin B-like activity regulation. In addition, the effect of E-64 on parasite survival and DNA fragmentation was tested. Results: Eight cathepsin B-like protease coding genes were identified in silico. Interestingly, while these sequences lacked the cathepsin B characteristic occluding loop, they maintained the catalytic active- site responsible for cathepsin B activity, which was evidenced by the increase in the degradation of the Z-RR-AMC substrate, suggesting the upregulation of the activity of these proteins. Additionally, inhibition of E-64 against G. duodenalis trophozoites caused a decrease in DNA fragmentation compared to control cells and had a positive effect on parasite survival after exposure to curcumin. Conclusion: Overall, these results suggested that Giardia duodenalis might have a cell death mechanism in which cathepsin B-like proteases play an important role.

Background: CIGB-300 is a pro-apoptotic peptide that abrogates CK2-mediated phosphorylation, and can elicit synergistic interaction in vitro and in vivo when combined with certain anticancer drugs. Objective: The combination of CIGB-300 with cisplatin is studied through data mining and expressionbased proteomics to reveal the molecular basis of this interaction. Cisplatin resistance-associated proteins, which have also been reported as CK2 substrates, were first identified by bioinformatic analyses. Methods: Data from these analyses suggested that the cisplatin resistance phenotype could be directly improved by inhibiting CK2 phosphorylation on specific substrates. Furthermore, 157 proteins were differentially modulated on the NCI-H125 lung cancer cell line in response to CIGB-300, cisplatin or both drugs as determined by LC-MS/MS. Results: The expression of 28 cisplatin resistance-associated proteins was changed when cisplatin was combined with CIGB-300. Overall, the proteins identified are also related to cell survival, cell proliferation and metastasis. Furthermore, the CIGB-300 regulated proteome revealed proteins that were initially involved in the mechanism of action of CIGB-300 and cisplatin as single agents. Conclusion: This is the first report describing the protein array modulated by combining CIGB-300 and cisplatin that will support the rationale for future clinical settings based on a multi-target cancer therapy.