Anita Horvatić

Recombinant human granulocyte colony stimulating factor pre-screening and screening of stabilizing carbohydrates and polyols.

Protein stabilization by solvent additives is frequently used concept in formulation development, although new technologies implemented over the past decade can improve protein biophysical as well as clinical properties by protein structural design (e.g. PEGylation, acylation, hesylation). The scope of this work was to evaluate the effect of chosen carbohydrate or polyol stabilizer in the formulation; firstly on linear peptide sequences on instable model of rHuG-CSF cleaved macromolecule by novel method named protein and peptide stabilizer pre-screening PPSP (formulated tryptic digest mixture stability evaluation in 54 h) and on overall stability of rHuG-CSF macromolecule by quantifying all relevant degradation parameters. Comprehensive protein stabilizing screening study included conformational analysis of formulated rHuG-CSF protein to obtain information on its secondary structure conformational stability. Protein aggregation induced by modulating conditions in solution (e.g. thermal stress and agitation) was monitored over discrete time periods. Oxidation and deamidation, as well as truncation or hydrolysis were accurately quantified. Together with pre-screening data, obtained by fast and resourceful amino acid sequence degradation analysis by LC-MS, statistical data evaluation of stabilizing contribution of substances selected from group of carbohydrates and polyols was performed. According to the statistical interpretation of obtained results the stabilizers were ranked in the following order: turanose, D-trehalose, lactitol, acetate buffer (non-stabilized sample), xylitol, cellobiitol, sorbitol, D-lyxose, leucrose, sorbitol without polysorbate, cellobiose.

Comparison between enhanced MALDI in-source decay by ammonium persulfate and N- or C-terminal derivatization methods for detailed peptide structure determination.

Amino acid sequencing and more detailed structure elucidation analysis of peptides and small proteins is a very difficult task even if state-of-the-art mass spectrometry (MS) is employed. To make this task easier, chemical derivatization methods of the N terminus with 4-sulfophenyl-isothiocyanate (SPITC) or the C terminus with 2-methoxy-4,5-dihydro-1H-imidazole (Lys-tag) can enhance peptide fragmentation or fragment ionizability, via proton mobility/localization mechanisms making tandem MS (MS(2)) spectra more informative and less demanding for structural interpretation. Observed disadvantages related to both derivatization methods are sample- and time-consuming procedures and the increased number of reaction byproducts. A novel, sulfate radical in-source formation method of matrix-assisted laser desorption ionization (MALDI) MS based on chemically enhanced in-source decay (ISD) can be accomplished by simple addition of ammonium persulfate (APS) in the matrix solution. This method enables effective decomposition of peptide ions already in the first stage of MS analysis where a large number of fragment ions are produced. The resultant MALDI-ISD mass spectra (MS after APS → MALDI-ISD MS) are almost equivalent to conventional, collision-induced dissociation (CID) MS(2) spectra. These fragment ions are further subjected to the second stage of the MS, and consequently, MS(3) spectra are produced, which makes the sequence analysis more informative and complete (CID MS(2) is thus equivalent to CID MS(3)). Multiply stage MS after APS addition showed enhanced sensitivity, resolution, and mass accuracy compared to peptide derivatization (SPITC and Lys-tag) or conventional MS and MS(2) analyses and offered more detailed insight into peptide structure.

Proteomic analysis of Mammillaria gracilis Pfeiff. in vitro-grown cultures exposed to iso-osmotic NaCl and mannitol

Salt and drought stress are important abiotic factors that negatively affect plant growth and productivity. Defense mechanisms, which plants have developed to cope with stress, are followed by alterations in a genome expression profile that in turn result in qualitative and quantitative change of the proteome. Although proteomic-based approach for studies of plant responses to salinity and drought has already been successfully employed in several plants, for cactus species such analyses have not been done so far. Therefore, in this study we have performed proteomic analysis of Mammillaria gracilis Pfeiff. in vitro-grown cultures, callus and tumor, exposed to iso-osmotic NaCl and mannitol. Obtained results differed among analyzed tissues. The higher number of differentially expressed proteins after either salt or mannitol treatment was revealed in tumor compared to callus. According to classification to different functional categories, majority of the identified callus responsive proteins belongs to protein synthesis and processing category, while the highest number of identified tumor proteins belongs to category of metabolism, which suggest that the mechanisms that mediate responses to salt- and mannitol-induced stress in cactus callus and tumor are dependent on tissue type. Down-regulation of proteins involved in cell protection suggests the inability of tumor to activate protective processes against salinity and osmotic stress.

Identification of novel biomarkers for treatment monitoring in canine leishmaniosis by high-resolution quantitative proteomic analysis

The objective of this study was to use the Tandem Mass Tag (TMT) isobaric label-based proteomic approach, in order to identify new potential biomarkers for the treatment monitoring of canine leishmaniosis that could not be identified by the use of gel-based techniques. For this purpose serum samples were obtained from 5 clinically diseased dogs before and one month after the treatment of canine leishmaniosis. The non-depleted serum samples were subjected to reduction, alkylation and trypsin digestion, and the resulting peptides were labeled using 6-plex TMT reagents. To obtain information about protein identities and relative quantification, liquid chromatography-MS analysis of multiplexed TMT-labeled peptides was employed. This gel-free, label-based quantitative proteomic approach enabled identification of 117 canine proteins. Among these, 23 showed significant difference (p<0.05) in expression (two downregulated and 21 upregulated ranging from 1.25 to 2.5 fold change). Comparison of gel-free TMT-based quantification and a gel-based approach previously applied to the same samples resulted in the identification of some common markers (Apo-A1, vitamin D binding protein and RBP4). However, 20 additional differentially represented proteins were highlighted by the gel-free approach, 13 of which have not been previously reported in canine leishmaniosis. In conclusion, the TMT-based proteomic approach allowed identification of new serum proteins that significantly change in concentration after canine leishmaniosis treatment. These proteins are involved in various physiopathological processes such as inflammatory, coagulation or defense mechanisms, and could potentially be suitable biomarkers for treatment monitoring of this parasitic disease.

Inflammatory Gene Expression Upon TGF-β1-Induced p38 Activation in Primary Dupuytren's Disease Fibroblasts

Objectives: Inflammation is an underlying mechanism behind fibrotic processes and differentiation of cells into myofibroblasts. Presented study therefore provides new data on activation of autoimmune and inflammatory immune response genes that accompany activation of p38 and cell differentiation in primary cells derived from Dupuytrens disease (DD) patients. Methods: Primary non-Dupuytrens disease cells (ND) were isolated from macroscopically unaffected palmar fascia adjacent to diseased tissue obtained from patients diagnosed with the last stage of DD and cultured in vitro. Gene expression, collagen gel contraction assay and analysis of secreted proteins were performed in ND cells treated with TGF-β1 and/or inhibitor of p38 phosphorylation. Results: During differentiation of ND fibroblasts, increased expression of immune response genes PAI-1, TIMP-1, CCL11, and IL-6 was found. These changes were accompanied by increased cell contractility and activation of p38 and its target kinase MK2. Inhibition of p38 phosphorylation reversed these processes in vitro. Conclusions: TGF-β1 induced p38 phosphorylation in ND cells grown from macroscopically unaffected palmar fascia adjacent to diseased tissue from DD patients. This was accompanied by activation of the cytokine genes CCL-11 and IL-6 and secretion of extracellular matrix regulatory proteins PAI-1 and TIMP-1. A combined approach directed toward inflammation and p38 MAPK-mediated processes in DD might be considered for improving management of DD patients and prevention of recurrence.

Proteomics in Veterinary Medicine and Animal Science: Neglected Scientific Opportunities with Immediate Impact

Animal/veterinary proteomics is an evolving field which holds a great promise not only for fundamental and applied discoveries regarding biology and pathology of domestic species, but can also be implemented in comparative applications of human diseases research. Experimental proteomics in domestic animals have advantages over use of rodents, such as multiple sampling in time series and availability of biological samples in sufficient volume for multiple analyses, such that both experimental and natural disease processes can be investigated. While there are certain technical limitations in the expansion of the field, they can currently be circumvented and in the future mastered with a greater participation of proteomic experts, which will in turn drive the accessibility of species‐specific reagents, data volume expansion in bioinformatic databases, and increased funding. This Viewpoint highlights some comparative proteomics studies addressing important issues and encourages readers to expand their horizons of domestic animal proteomics research. It will hopefully inspire new fruitful collaborations between veterinary and animal scientists and proteomic specialists for research in these areas that can have immediate and direct impact on health, society, and the economy.

Antitumor mechanisms of amino acid hydroxyurea derivatives in the metastatic colon cancer model

The paper presents a detailed study of the biological effects of two amino acid hydroxyurea derivatives that showed selective antiproliferative effects in vitro on the growth of human tumor cell line SW620. Tested compounds induced cell cycle perturbations and apoptosis. Proteins were identified by proteomics analyses using two-dimensional gel electrophoresis coupled to mass spectrometry, which provided a complete insight into the most probable mechanism of action on the protein level. Molecular targets for tested compounds were analyzed by cheminformatics tools. Zinc-dependent histone deacetylases were identified as potential targets responsible for the observed antiproliferative effect.

Surface Proteome Biotinylation Combined with Bioinformatic Tools as a Strategy for Predicting Pathogen Interacting Proteins

Constant advancements in methodology and mass spectrometry instrumentation, genome sequencing and bioinformatic tools have enabled the identification of numerous pathogen proteomes. Identifying the pathogen interacting proteins by means of high-throughput techniques is key for understanding pathogen invasion and survival mechanisms and in such a way proposing specific proteins as pharmaceutical targets. Herein we describe the methodology for the enrichment and identification of pathogen surface proteome using cell surface protein biotinylation followed by LC-MS/MS and bioinformatic analyses of such data. This strategy is to be employed for the determination of protein subcellular localization and prediction of potential pathogen interacting proteins.

Quantitative proteomics using tandem mass tags in relation to the acute phase protein response in chicken challenged with Escherichia coli lipopolysaccharide endotoxin

The inflammatory response in chickens (Gallus Gallus domesticus) is an integral part of the birds response to infection. Detailing proteomic changes occurring during infection would be beneficial to the poultry industry, offering opportunities for comparative pathophysiological analysis. The objective of this study was to quantify the changes in the plasma proteome in chickens challenged with lipopolysaccharide (LPS), a bacterial endotoxin known to stimulate the host innate immune system. Plasma from chicken (N = 6) challenged with Escherichia coli (LPS) (2 mg/kg body weight) was collected pre (0 h) and at 12, 24, 48, and 72 h post-injection along with plasma from a control group (N = 6) challenged with sterile saline. Samples were analysed by a quantitative Tandem Mass Tags approach using a Q-Exactive-Plus mass-spectrometer. Identification and relative quantification were performed using Proteome Discoverer, and data were analysed using R. Gene Ontology terms were analysed by Cytoscape based on the Gallus gallus database. Finally, 87 significantly regulated proteins were found, including serum-amyloid-A, ovotransferrin and alpha-1-acid-glycoprotein, showing a significant effect of time post-injection in the LPS-treated group. Different pathways related with protein activation cascade and heterotopic cell-cell adhesion were affected by LPS-challenge. LPS-challenged chickens demonstrate significant changes to the plasma proteome with both increases and decreases of individual proteins within 12 h of challenge. SIGNIFICANCE: The injection of chicken with bacterial lipopolysaccharide followed by sequential plasma and clinical analysis of the bird, is a long established and a widely used model for inflammation and infection studies. This study, utilising and combining proteomic and immunoassay analysis with bioinformatic analysis, revealed that several biological pathways are modulated during this early period of inflammation. In addition, proteins with biomarker potential were identified and successfully validated. This experimental model also demonstrated potential for pathophysiological mechanism investigation and as an inflammatory model for biomedical research. There is, despite plasma being an easily accessible biological matrix which is representative of the health status of the bird, scarce data on the chicken plasma proteome. This research makes a positive contribution to the current field, generating significant data for continuing comparative analysis.

Integrated dataset on acute phase protein response in chicken challenged with Escherichia coli lipopolysaccharide endotoxin

Data herein describe the quantitative changes in the plasma proteome in chickens challenged with lipopolysaccharide (LPS), a bacterial endotoxin known to stimulate the host innate immune system obtained by shotgun quantitative proteomic tandem mass tags approach using high-resolution Orbitrap technology. Statistical and bioinformatic analyses were performed to specify the effect of bacterial endotoxin. Plasma from chicken (N=6) challenged with Escherichia coli (LPS) (2 mg/kg body weight) was collected pre (0 h) and at 12, 24, 48, and 72 h post injection along with plasma from a control group (N=6) challenged with sterile saline. Protein identification and relative quantification were performed using Proteome Discoverer, and data were analysed using R. Gene Ontology terms were analysed by the Cytoscape application ClueGO based on Gallus gallus GO Biological Process database, and refined by REVIGO. Absolute quantification of several acute phase proteins, e.g. alpha-1-acid glycoprotein (AGP), serum amyloid A (SAA) and ovotrensferrin (OVT) was performed by immunoassays to validate the LC-MS results. The data contained within this article are directly related to our research article”Quantitative proteomics using tandem mass tags in relation to the acute phase protein response in chicken challenged with Escherichia coli lipopolysaccharide endotoxin” [1]. The raw mass spectrometric data generated in this study were deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD009399 (http://proteomecentral.proteomexchange.org/cgi/GetDataset?ID=PXD009399).