Changes of serum protein N-glycosylation in cancer.

BACKGROUND: Glycosylation is a posttranslational modification responsible for many bio-logical processes including protein-protein interactions, cell signaling or cell cycle regulation. Changes in glycosylation of serum proteins reflects the status of tissues and cells in the organism and therefore can be used as markers for dia-gnosis of cancer, its progression and determination of its subtypes. N-glycan profiling is often used for characterization of N-glycosylation changes. It is based on the measurements of N-glycans released from the serum proteins. Beside the N-glycan profiling, glycoproteomic approach is emerging as it preserves the information about glycan composition, original protein, and its glycosylation sites. PURPOSE: This review covers existing works describing the changes in serum protein N-glycosylation in various cancer types. Attention was paid to both the glycomic and glycoproteomic approaches. The last part of the review shortly presents the analytical methods used for these analyses.

Glycoproteins in the Sera of Oncological Patients.

BACKGROUND: Glycosylation is a posttranslational modification that is involved in many biological processes and significantly affects the processes associated with tumour progression. Changes in glycan structures on the surface of tumour cells caused by altering levels of glycosyltransferase and glycosidase expression affect proliferation, adhesion, migration and cellular signalling. The presence of aberrant glycan structures and glycoconjugates in the sera of oncological patients has been reported in many cancers. Consequently, many glycoproteins have been approved by the U.S. Food and Drug Administration as tumour biomarkers for clinical investigations. At present, attention is focused on the search for new glycomarkers that are decorated by aberrant glycosylation or are overexpressed in the serum or exosomes due to their active secretion or release from tumour cells to the extracellular space. PURPOSE: The aim of this article has been to review the structure of glycans, glycoproteins and other glycoconjugates and to give more details about their functions in the development and progression of tumours. Another aim was to familiarise the reader with selected clinically approved glycoproteins used to diagnose oncological diseases (AFP, PSA, CA 125, HE4). Attention was paid to changes in the glycan structure of these proteins, their function, serum concentrations and clinical use in the diagnostics of cancer.

Glycosylation as an Important Regulator of Antibody Function.

BACKGROUND: The glycosylation of constant regions of antibodies significantly affects their interaction capabilities with immune cells. It is a modification that, in addition to the bio-logical activity of antibodies, has an impact on their conformation, stability, solubility, secretion, pharmaco-kinetics, and immunogenicity. The location of glycosylations on the molecule is essential for the proper function of the antibody, as is the structure of the individual glycans. Changes in the glycosylation profiles of antibodies have been described in some physiological processes like pregnancy or ageing, but also in many pathological conditions such as rheumatoid arthritis or gastric, lung and prostate tumours. There are still several unexplained mechanisms that control the glycosylation of antibodies or immune responses, which in turn are regulated by these modifications. Multiple sources describe the importance of some specific glycosylations as potential bio-markers. PURPOSE: The aim of this review is to summarise and present the knowledge of the glycosylation of antibodies and to highlight their influence on immune responses and their role dur-ing dis-ease. Their importance is also underlined by the fact that the most of these therapeutic antibodies used and developed are modified by glycosylation. The targeted introduction of appropriate glycosylations, which can promote activities such as antibody--dependent cellular cytotoxicity, antibody--dependent cellular phagocytosis or complement--dependent cytotoxicity, have improved the ability of these antibodies to kill pathogens or tumour cells. Therefore, more attention is be-ing paid to this area. In the future, more effective tools for dia-gnos-ing and treat-ing certain dis-eases can be created with better knowledge.

Protein Ubiquitination Research in Oncology.

BACKGROUND: Ubiquitination is a vital posttranslational protein modification involved in the regulation of many eukaryotic signalling pathways. Aberrant ubiquitin signalling is known to be a molecular causality of certain cancer, neurodegenerative, immune system or cardiovascular diseases. The recent development of mass spectrometry methods enables qualitative and quantitative ubiquitination analysis in biological material from cancer patients. Research of ubiquitination may clarify the molecular cause of aberrant changes in the protein level of tumour suppressors or oncogenes. PURPOSE: We aim to explain the meaning and importance of ubiquitination in certain molecular processes taking place in the human body. We hereby emphasise the connection between ubiquitination and malignant processes. A literature search is followed by introducing our mass spectrometry platform intended for ubiquitin identification via diglycyl remnants in the CHIP protein sequence. The aim is to introduce tandem mass spectrometry identification of ubiquitin modification, ubiquitination tandem mass spectra validation and the time-dependent manner of CHIP ubiquitination to the reader. CONCLUSION: A literature search familiarises the reader with known mechanisms of aberrant ubiquitination in malignant diseases. A successfully optimised mass spectrometry platform could serve as a potent tool for determining ubiquitin position in proteins that are a part of real tumour samples.

[Ascites May Provide Useful Information for Diagnosis of Ovarian Cancer]. / Ascitická tekutina u nádoru vajecníku muze poskytnout informace vhodné pro diagnostiku.

BACKGROUND: Ovarian cancer is the most lethal gynecological cancer, almost 80% of all patients succumb the disease within 5 years of diagnosis. High mortality is caused especially by nonspecific symptoms, diagnosis in late stages and the absence of a specific biomarker. Currently, the most common diagnostic biomarkers are the membrane glycoprotein Cancer Antigen 125 (CA 125), the Human Epididymal Protein 4 (HE4) and the Carcinoembryonic Antigen (CAE). None of these biomarkers is specific only for ovarian cancer and increased levels may be caused by other diseases. Therefore, current research is focused on finding new biomarkers for diagnosis and prognosis of ovarian cancer. Interesting clinical material is ascites, the fluid accumulated in abdominal cavity, which is typical for ovarian cancer and it is present in almost 90% of all cases of stage III and IV. MATERIAL AND METHODS: For this study, samples of ascites from patients with benign and malignant ovarian tumors were used. For full glycomic and proteomic analysis, only 5 µL of ascites were used. Glycans were released from proteins by the enzyme PNGase F and proteins were digested to peptides by trypsin. Samples were purified and measured using a mass spectrometer. RESULTS: Glycan and protein profiles of patients with benign and malignant ovarian cancer were compared. In patient with a benign tumor, more simple glycans with lowm/z were increased while in the patient with a malignant tumor, higher, more complex glycans were increased. In the malignat tumor in comparison to benign tumor, 127 unique proteins were identified, especially proteins of the annexin, mucin and peroxiredoxin families. CONCLUSION: This investigation is a pilot study focused on comparison of protein and glycan composition of ascites in patients with benign and malignant ovarian cancer. Significant differences were found on both glycan and protein levels. Results will be verified on a larger set of patients and compared with a set of control samples.Key words: glycomics - proteomics - ascitic fluid - ovarian cancer This study was supported by projects of the Ministry of Education Youth and Sports - National Sustainability Program I - LO1413; Ministry of Health, Czech Republic - conceptual development of research organization (MMCI, 00209805); Czech Science Foundation 16-04496S. The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers.Submitted: 13. 3. 2017Accepted: 26. 3. 2017.

[Utilization of Hydrogen/Deuterium Exchange in Biopharmaceutical Industry]. / Vyuzití metody vodík/deuteriové výmeny v biofarmaceutickém prumyslu.

BACKGROUND: The development of biopharmaceutics is the fastest growing segment of the present pharmaceutical industry. The analysis of proteins therapeutics is a challenging task due to their large size and complexity of spatial structure. Any changes in the primary, secondary, tertiary or quaternary protein structure can have huge impact on their function, efficiency and toxicity. Mass spectrometry proved itself to be a powerful tool for analysis of primary protein structure (amino acid sequence) and thanks to the development of new techniques in last years it is able to analyse higher order protein structures. One of these new techniques is hydrogen/deuterium exchange (HDX). HDX is based on exchange of amid protons with deuterium from solution on the protein backbone chain. Protons on the surface of protein are exchanging with deuterium much faster than protons buried inside of protein. HDX results could provide information about spatial protein structure and also about protein-protein interactions and protein-ligand interactions. Furthermore, by analysing of deuterium exchange in different time points this method could give information about dynamic changes of protein structure and dynamics of proteins interactions. Because of possibilities of this method, HDX become attractive method for characterization of protein biopharmaceuticals. AIMS: This review article is focused on the utilization of mass spectrometry in biopharmaceutical industry and mainly on HDX method and its applications.Key words: mass spectrometry - proteomics - protein conformation - drug discovery - drug industry - hydrogen/deuterium exchangeThe work was supported by the project MEYS - NPS I - LO1413.The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study.The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers.Submitted: 23. 5. 2016Accepted: 10. 6. 2016.

[New Technologies for In Vivo Cancer Diagnostics]. / Nové technologie pouzívané pro in vivo diagnostiku nádoru.

BACKGROUND: The treatment of oncological diseases is based on the combination of surgery, representing the key step for the removal of the tumor tissue, radiotherapy, chemotherapy, and hormone therapy. However, the surgery is often accompanied by issue of determining the boundaries of the tumor. Prior the operation, the surgeon has information on preoperative findings, which indicate the location and extent of the tumor, but does not specify a clear boundary between the tumor and healthy tissue. This area cannot be recognized visually or by touch in most cases and when the tumor is not removed completely the patient has to undergo reoperation. AIM: Therefore, a number of research centers began to deal with the development of technology that would provide information about the state of the tissue in real time directly during surgery and would not require the collection or storage of tissue samples. These include MarginProbe, Spectropen tissue and spectroscopic scanner devices. Another group consists of imaging techniques using mass spectrometry approaches to determine the tissue specificity. Recently, the intraoperative mass spectrometry (REIMS) technique has undergone tremendous development. It uses an electronic scalpel using by the surgeon for cutting the tissue, when the resulting aerosol is discharged into the mass spectrometer that in tenths of seconds measures mass spectra of phospholipids, which are specific to the operated tissue (tumor or healthy). In the Czech Republic this technology has been already used for research purposes for the detection of drug deposited in the tumor and healthy tissue of mice suffering from melanoma. The obtained results show that with this apparatus it would be possible fundamentally affect the treatment and its efficacy in oncology as well. We will inform you about these new technologies and elucidate their principles and utilization.Key words: surgery - cancer - tumors - molecular diagnostics - mass spectrometry - databaseThis work was supported by the project MEYS - NPS I - LO1413.The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study.The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers.Submitted: 17. 5. 2016Accepted: 6. 9. 2016.

Recombinant expression, in vitro refolding and characterizing disulfide bonds of a mouse inhibitory C-type lectin-like receptor Nkrp1b.

As a part of the innate immunity, NK (Natural Killer) cells provide an early immune response to different stimuli, e.g. viral infections and tumor growths. However, their functions are more complex; they play an important role in reproduction, alloimmunity, autoimmunity and allergic diseases. NK cell activities require an intricate system of regulation that is ensured by many different receptors on a cell surface which integrate signals from interacting cells and soluble factors. One way to understand NK cell biology is through the structure of NK receptors, which can reveal ligand binding conditions. We present a modified protocol for recombinant expression in Escherichia coli and in vitro refolding of the ligand-binding domain of the inhibitory Nkrp1b (SJL/J) protein. Nkrp1b identity and folding was confirmed using mass spectrometry (accurate mass of the intact protein and evaluation of disulfide bonds) and one-dimensional nuclear magnetic resonance spectroscopy. The intention is to provide the basis for conducting structural studies of the inhibitory Nkrp1b protein, since only the activating Nkrp1a receptor structure is known.

[What Can Study of Oligomerization of Proteins in the Process of Oncogenesis Bring Us?]. / Co muze prinést studium oligomerizace proteinu v procesu onkogeneze?

Many cellular proteins form oligomers. The equilibrium between monomeric and oligomeric states of these proteins is important for the regulation of protein activity. Modulation of the oligomerization equilibrium could be an interesting approach in the development of new therapeutic agents. This review summarizes information about protein oligomerization and modulation of this process, demonstrating the role of oligomerization in oncogenesis by tumor suppressor protein p53, which forms tetrameric structure. Today, many studies focus on finding compounds that stabilize its tetramers. Among the methods for studying oligomerization, we present hydrogen/ deuterium exchange method coupled with mass spectrometry which is suitable for the detection of protein-protein interaction and analysis of oligomerization dynamics.

[Sugars Interfere or Glycomics in the Field of Cancer Biomarkers]. / "Cukry zasahují" aneb glykomika na poli nádorových biomarkeru.

Glycomics is concerned with detection and characterization of glycans present in biological samples. It is well-known that glycan structures impart high degree of structural diversity to biomolecules and thus add wide -ranging biological functions, such as cellular recognition, adhesion or involvement in cellular signaling pathways. They substantially participate in oncogenesis, e. g. in phases of invasion, metastasis and angiogenesis. Therefore, analysis of glycan structures in tumor tissues or body liquids is a promising tool for searching for potential tumor biomarkers essential for an early diagnosis of the neoplastic disease. The presented review describes the process of glycosylation and the origination of N  and O  glycans, presenting examples of glycan profiling in pancreatic, prostate and ovarian cancer.

[Analysis of Phosphoproteome Changes in MDA MB 468 Cancer Cell Line in Response to Expression of p63 Isoforms Using Mass Spectrometry]. / Analýza zmen fosfoproteomu nádorové bunecné linie MDA MB 468 v odpovedi na expresi izoforem p63 pomocí hmotnostní spektrometrie.

Compared to normal cells, tumor cells can show different activity of kinases and phosphatases resulting in altered phosphorylation states of proteins affecting their activity within various signaling pathways. The detection of these alterations is essential for development of targeted therapy based on activation/ inhibition of specific signaling pathways. Various methods can be used for detection of protein phosphorylation; however, a comprehensive assessment of phosphoproteome is performed by mass spectrometry. The differences in phosphoproteome were studied using MDA  MB  468 cell line (with incorporated genes encoding isoforms of p63) derived from breast carcinoma. Cells with tetracycline-induced expression of the p63 isoforms were compared to control cells with wildtype expression. Denatured proteins from cell lysates were digested to peptides, enriched for phosphopeptides and subsequently separated using liquid chromatograph coupled with mass spectrometer Orbitrap Elite. Three different mass spectrometric methods were used for each sample analysis to find the most suitable conditions for the detection of phosphorylated peptides. Then phosphoproteins were identified and quantified. The number of identified phosphoproteins using all chosen mass spectrometric methods was similar; however, each method showed several unique phosphorylated proteins. Our analysis revealed that both p63 isoforms (TAp63α a Np63α) mainly affected phosphorylation of proteins associated with RNA splicing in MDA- MB- 468 cells.

[Adenoviral Vectors in Gene Therapy]. / Adenovírusové vektory v génovej terapii.

This review is focused on gene therapy, especially adenovirus vectors and their relationship with the immune system response. Adenovirus vectors belong to the most used gene delivery vehicles in gene therapy, study of gene expression or immunotherapy. One of the most important questions concerning their use is their influence on organism in vivo. Study of immunomodulating properties of the adenovirus vectors opens a way for further manipulation and their more effective practical use.

[Quantitative mass spectrometry and its utilization in oncology]. / Kvantitativní hmotnostní spektrometrie a její vyuzití v onkologii.

Cancers are genetically and clinically very heterogeneous diseases; therefore, various proteomic studies have been trying to find bio-markers which can facilitate prognosis, diagnosis or treatment of these oncological diseases. The mass spectrometry is an effective tool for identification, quantitation, and characterization of biomolecules in the complex bio-logical samples. The first step suitable for selection of bio-markers called discovery proteomics provides a detailed analysis of the samples contributing to the identification of proteins, comparison of their presence in the samples, and selection of the convenient candidates for the prospective bio-markers. The next step of proteomics analysis is directed towards verification of chosen bio-markers with the approach called targeted proteomics. This technique evaluates presence and quantity of the proteins (biomarkers) in clinically precisely defined samples. This article focuses on the description of various approaches suitable for the quantitative analysis of the proteins connected with mass spectrometry.

[Analysis of protein using mass spectrometry]. / Analýza proteinu pomocí hmotnostní spektrometrie.

Recently, mass spectrometry has become a powerful tool in cancer research. Mass spectrometry represents the method that allows identification, quantification and characterization of proteins in bio-logical samples. Nowadays, it is mainly used for bio-marker discovery that can enable early detection of cancer. This article is focused on protein analysis by mass spectrometry. At first, mass spectrometry and its importance in proteomics are described. Subsequently ionization type and mass analyzers are discussed. This relates to the possibility of online or offline analysis connection with separation techniques, such as liquid chromatography and electrophoresis. Different approaches for preparing proteins and methods of analysis of bio-molecules using mass spectrometers are described. In addition, the possibility of mass spectrometric analyses of samples and data processing are discussed.

[New trends in the study of protein glycosylation in oncological diseases]. / Nové trendy ve studiu glykosylace proteinu u onkologických onemocnení

Glycomics and glycoproteomics represent relatively new directions in detail analyses of complex bio-logical media. These areas of increasing importance to cancer research complement the more established genomic profiling and proteomics. Glycoproteins are being increasingly recognized as important in cellular interactions and adhesion. Structural alterations of their glycan moieties seem to occur in different cancer conditions. We review current directions in glycomic profiling and glycoproteomic investigations of bio-logical fluids and tissues pertaining to cancer. The used methods rely on capillary separation techniques, mass spectrometry, and the glycan and lectin arrays. They all show considerable promise for new diagnostic and prognostic measurements.

[Ananlysis of phosphoproteins and signalling pathways by quantitative proteomics]. / Analýza fosfoproteínov a signálnych dráh kvantitatívno proteomickými metódami.

Protein phosphorylation is a key regulator in cellular signaling pathways. It is involved in most cellular events in which interplay between phosphatases and kinases strictly controls bio-logical processes, such as differentiation, proliferation and apoptosis. Altered or defective signaling pathways often result in various diseases, emphasizing the importance of studying the phosphoproteome. The abundance of phosphoproteins in the proteome is often very low, which requires specific and highly sensitive approaches. By using quantitative proteomics methods, we are able to analyze changes in abundance of proteins and their posttranslational modifications and then changes in signaling pathways. In this review, we describe quantitative proteomics methods, which could be used for study of phosphoproteins and their connection in signaling pathways.

A combined immunoprecipitation and mass spectrometric approach to determine deltaNp63-interacting partners.

Expression of p63 is essential for the formation of epidermis and other stratifying epithelia. Moreover p63 is highly expressed in several epithelial cancers and is involved in tumourigenesis and controlling chemo-sensitivity. The identification of p63 interacting partners is essential for understanding the complex network of gene regulation managing epithelial development and could also help to reveal signalling pathways participating in UV-damage response in human skin. We used a proteomic approach to identify proteins that interact with deltaNp63. Proteins were isolated by immunoprecipitation with deltaNp63 specific antibody and analysed by mass spectrometry. We identified 23 proteins as potential deltaNp63 binding partners that were not present in negative control samples. These results will be evaluated using other methods.

The problems of proteinuria measurement in urine with presence of Bence Jones protein.

OBJECTIVES: Protein concentration measurement in the urine can be problematic in the presence of Bence Jones protein. We have carried out an external quality control assessment with the participation of 79 clinical biochemistry laboratories from the Czech Republic and Slovakia. DESIGN AND METHODS: The laboratories received a reference urine sample obtained from a patient with multiple myeloma and lambda free light chain proteinuria and were asked to type the paraprotein using immunofixation and to measure total urinary protein using their established method, most commonly turbidimetry, pyrogallol red assay, and biuret assay. RESULTS: There was a very wide inter-laboratory variability in the protein concentration readouts with up to three-fold difference in some cases. High-resolution two-dimensional electrophoresis and linear mass spectrometry showed that a high proportion of the urinary paraprotein was composed of lambda light chain fragments with molecular weight of 12kDa. CONCLUSIONS: Our results highlight the challenges of reliable and reproducible measurement of urinary protein concentration in the presence of Bence Jones protein.

Identification of alphaB-crystallin, a biomarker of renal cell carcinoma by SELDI-TOF MS.

Spectrometric-based surface-enhanced laser desorption/ionization ProteinChip (SELDI-TOF) facilitates rapid and easy analysis of protein mixtures and is often exploited to define potential diagnostic markers from sera. However, SELDI- TOF is a relatively insensitive technique and unable to detect circulating proteins at low levels even if they are differentially expressed in cancer patients. Therefore, we applied this technology to study tissues from renal cell carcinomas (RCC) in comparison to healthy controls. We found that different biomarkers are identified from tissues than those previously identified in serum, and that serum markers are often not produced by the tumors themselves at detectable levels, reflecting the nonspecific nature of many circulating biomarkers. We detected and characterized áB-crystallin as an overexpressed protein in RCC tissues and showed differential expression by immunohistochemistry. We conclude that SELDI-TOF is more useful for the identification of biomarkers that are synthesized by diseased tissues than for the identification of serum biomarkers and identifies a separate set of markers. We suggest that SELDI-TOF should be used to screen human cancer tissues to identify potential tissue-specific proteins and simpler and more sensitive techniques can then be applied to determine their validity as biomarkers in biological fluids.

Detection of specific spectral markers of Coxiella burnetii isolates by MALDI-TOF mass spectrometry.

Specific markers for Coxiella burnetii (C.b.) isolates RSA 493, Priscilla, and BUD were detected using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). The method revealed noticeable differences in the ion signal profiles of the isolates in the mass range of 318 kDa. The number of characteristic ions for RSA 493, BUD, and Priscilla was 24, 15, and 7, respectively. The specific markers were compared against C.b. database using the Tag-Ident proteomics tool. For the isolates RSA 493, Priscilla and BUD there were identified 11, 5 and 3 potential biomarkers, respectively. This method represents a powerful tool for the rapid, sensitive, and differential characterization of C.b. isolates and is a good candidate for phyloproteomic approaches.