Djuro Josić

High throughput isolation and glycosylation analysis of IgG - variability and heritability of the IgG glycome in three isolated human populations

All immunoglobulin G molecules carry N-glycans, which modulate their biological activity. Changes in N-glycosylation of IgG associate with various diseases and affect the activity of therapeutic antibodies and intravenous immunoglobulins. We have developed a novel 96-well protein G monolithic plate and used it to rapidly isolate IgG from plasma of 2298 individuals from three isolated human populations. N-glycans were released by PNGase F, labeled with 2-aminobenzamide and analyzed by hydrophilic interaction chromatography with fluorescence detection. The majority of the structural features of the IgG glycome were consistent with previous studies, but sialylation was somewhat higher than reported previously. Sialylation was particularly prominent in core fucosylated glycans containing two galactose residues and bisecting GlcNAc where median sialylation level was nearly 80%. Very high variability between individuals was observed, approximately three times higher than in the total plasma glycome. For example, neutral IgG glycans without core fucose varied between 1.3 and 19%, a difference that significantly affects the effector functions of natural antibodies, predisposing or protecting individuals from particular diseases. Heritability of IgG glycans was generally between 30 and 50%. The individuals age was associated with a significant decrease in galactose and increase of bisecting GlcNAc, whereas other functional elements of IgG glycosylation did not change much with age. Gender was not an important predictor for any IgG glycan. An important observation is that competition between glycosyltransferases, which occurs in vitro, did not appear to be relevant in vivo, indicating that the final glycan structures are not a simple result of competing enzymatic activities, but a carefully regulated outcome designed to meet the prevailing physiological needs.

Secretome analysis of an osteogenic prostate tumor identifies complex signaling networks mediating cross-talk of cancer and stromal cells within the tumor microenvironment

A distinct feature of human prostate cancer (PCa) is the development of osteoblastic (bone-forming) bone metastases. Metastatic growth in the bone is supported by factors secreted by PCa cells that activate signaling networks in the tumor microenvironment that augment tumor growth. To better understand these signaling networks and identify potential targets for therapy of bone metastases, we characterized the secretome of a patient-derived xenograft, MDA-PCa-118b (PCa-118b), generated from osteoblastic bone lesion. PCa-118b induces osteoblastic tumors when implanted either in mouse femurs or subcutaneously. To study signaling molecules critical to these unique tumor/microenvironment-mediated events, we performed mass spectrometry on conditioned media of isolated PCa-118b tumor cells, and identified 26 secretory proteins, such as TGF-β2, GDF15, FGF3, FGF19, CXCL1, galectins, and β2-microglobulin, which represent both novel and previously published secreted proteins. RT-PCR using human versus mouse-specific primers showed that TGFβ2, GDF15, FGF3, FGF19, and CXCL1 were secreted from PCa-118b cells. TGFβ2, GDF15, FGF3, and FGF19 function as both autocrine and paracrine factors on tumor cells and stromal cells, that is, endothelial cells and osteoblasts. In contrast, CXCL1 functions as a paracrine factor through the CXCR2 receptor expressed on endothelial cells and osteoblasts. Thus, our study reveals a complex PCa bone metastasis secretome with paracrine and autocrine signaling functions that mediate cross-talk among multiple cell types within the tumor microenvironment.

Synthesis, characterisation and in vitro investigation of photodynamic activity of 5-(4-octadecanamidophenyl)-10,15,20-tris(N- methylpyridinium-3-yl)porphyrin trichloride on HeLa cells using low light fluence rate

Photodynamic therapy (PDT) is a treatment that aims to kill cancer cells by reactive oxygen species, mainly singlet oxygen, produced through light activation of a photosensitiser (PS). Amongst photosensitisers that attracted the most attention in the last decade are cationic and amphiphilic molecules based on porphyrin, chlorin and phthalocyanine structures. Our aim was to join this search for more optimal balance of the lipophilic and hydrophilic moieties in a PS. A new amphiphilic porphyrin, 5-(4-octadecanamidophenyl)-10,15,20-tris(N-methylpyridinium-3-yl)porphyrin trichloride (5) was synthesised and characterised by (1)H NMR, UV-vis and fluorescence spectroscopy, and by MALDI-TOF/TOF spectrometry. In vitro photodynamic activity of 5 was evaluated on HeLa cell lines and compared to the activity of the hydrophilic 5-(4-acetamidophenyl)-10,15,20-tris(N-methylpyridinium-3-yl)porphyrin trichloride (7). Low fluence rate (2mWcm(-2)) of red light (643nm) was used for the activation, and both porphyrins showed a drug dose-response as well as a light dose-response relationship, but the amphiphilic porphyrin was presented with significantly lower IC50 values. The obtained IC50 values for 5 were 1.4μM at 15min irradiation time and 0.7μM when the time of irradiation was 30min, while for 7 these values were 37 and 6 times higher, respectively. These results confirm the importance of the lipophilic component in a PS and show a potential for 5 to be used as a PS in PDT applications.

Chapter Six – Use of Foodomics for Control of Food Processing and Assessing of Food Safety

Food chain, food safety, and food-processing sectors face new challenges due to globalization of food chain and changes in the modern consumer preferences. In addition, gradually increasing microbial resistance, changes in climate, and human errors in food handling remain a pending barrier for the efficient global food safety management. Consequently, a need for development, validation, and implementation of rapid, sensitive, and accurate methods for assessment of food safety often termed as foodomics methods is required. Even though, the growing role of these high-throughput foodomic methods based on genomic, transcriptomic, proteomic, and metabolomic techniques has yet to be completely acknowledged by the regulatory agencies and bodies. The sensitivity and accuracy of these methods are superior to previously used standard analytical procedures and new methods are suitable to address a number of novel requirements posed by the food production sector and global food market.

Chapter 29 - Detection of microbial toxins by – omics methods: a growing role of proteomics

Abstract Foodborne diseases caused by bacteria and their endotoxins and exotoxins hold enormous relevance for the public health sector, in particular in the context of the ever-growing globalization where proper monitoring and risk management approaches are still missing. Therefore, detection of bacteria and their exotoxins in food has been increasingly tackled by novel approaches and methods known as “-omics” methods. Bacterial genome sequencing proved to be the most mature technological platform for routine surveillance of microbiological risk assessment, while proteomics tools are increasingly used for fast bacterial subspecies differentiation, analysis of protein secretome, or as alternatives to immunoassay. In particular, mass spectrometry-based techniques showed a superior performance in descriptive and quantitative applications and methods combining liquid chromatography to mass spectrometry (LC-MS and LC-MS/MS) are constantly developed for increased proteome coverage. Still, initial high investments in mass spectrometry instrumentation and open issues in protocol optimization seems to be major bottlenecks for broader use of this technique in routine food analyses.Foodborne diseases caused by bacteria and their endotoxins and exotoxins hold enormous relevance for the public health sector, in particular in the context of the ever-growing globalization where proper monitoring and risk management approaches are still missing. Therefore, detection of bacteria and their exotoxins in food has been increasingly tackled by novel approaches and methods known as “-omics” methods. Bacterial genome sequencing proved to be the most mature technological platform for routine surveillance of microbiological risk assessment, while proteomics tools are increasingly used for fast bacterial subspecies differentiation, analysis of protein secretome, or as alternatives to immunoassay. In particular, mass spectrometry-based techniques showed a superior performance in descriptive and quantitative applications and methods combining liquid chromatography to mass spectrometry (LC-MS and LC-MS/MS) are constantly developed for increased proteome coverage. Still, initial high investments in mass spectrometry instrumentation and open issues in protocol optimization seems to be major bottlenecks for broader use of this technique in routine food analyses.

Plasma biomarker identification in S-adenosylhomocysteine hydrolase deficiency

S‐Adenosylhomocysteine hydrolase (AHCY) deficiency is a rare congenital disorder in methionine metabolism clinically characterized by white matter atrophy, delayed myelination, slowly progressive myopathy, retarded psychomotor development and mildly active chronic hepatitis. In the present study, we utilized a comparative proteomics strategy based on 2‐DE/MALDI‐MS and LC/ESI‐MS to analyze plasma proteins from three AHCY‐deficient patients prior to and after receiving dietary treatment designed to alleviate disease symptoms. Obtained results revealed candidate biomarkers for the detection of myopathy specifically associated with AHCY deficiency, such as carbonic anhydrase 3, creatine kinase, and thrombospondin 4. Several proteins mediating T‐cell activation and function were identified as well, including attractin and diacylglycerol kinase α. Further validation and functional analysis of identified proteins with clinical value would ensure that these biomarkers make their way into routine diagnosis and management of AHCY deficiency.

Chapter 5 – Protein and peptide separations

Abstract Since the introduction of suitable, highly porous supports and later nonporous and monolithic supports, chromatography has been an indispensibleindispensable method for protein separations on the analytical and preparative scales. The most frequently used methods for protein chromatography are ion exchange, reversed- phase, hydrophobic hydrophobic-interaction chromatography, chromatography on hydroxyapatite and different types of affinity, and pseudo-affinity chromatography. Because of the use of organic solvents during the separation in reversed-phase chromatography, denaturation and loss of biological activity frequently occurs, and this method is less suitable for the separation of biologically active, therapeutic proteins. Chromatography on monolithic supports and chromatography in the displacement mode offer additional opportunities for fast, highly effective separation of proteins on both the analytical and preparative scales.

The Role of Proteomics in Personalized Medicine

The consequences of the differences in the genome of each human individual are the variation in expressed protein isoforms, as well as the changes of the level and timing of protein expression and of the spatial distribution of expressed proteins. These differences are defined as differences in human proteome. The consequence of the changes of individual proteome is the difference in protein–protein interactions (interactome). The network of interactions between proteins underlines every single process in a living organism and makes it happen. This complex network is kept in more or less optimal homeostasis that is unique for each organism. Thus, the capacity of the interactome to overcome disease condition differs among individuals, e.g., to compensate over or down regulations of different processes that are induced, e.g., by mutations. Recent technological advances in high throughput proteomic techniques enabled fast and deep analysis of human proteome and also some understanding of the complex mechanism of protein–protein interactions. However, the human proteome analysis is still not complete, and further improvements in both analytical techniques and accompanying bioinformatics tools are necessary. In this chapter, current state of application of different proteomic approaches for personalized patient proteome profiling and search for diagnostic and prognostic disease biomarkers are presented and the contribution of proteome analysis to personalized approach in most frequent diseases in developed Western World, namely cancer, cardiovascular, urological and neurodegenerative diseases, diabetes mellitus and allergies, has been reviewed.