[Building Mass Spectrometry Spectral Libraries of Human Cancer Cell Lines]. / Tvorba hmotnostne spektrometrických spektrálnych knizníc nádorových bunecných línií.

BACKGROUND: Cancer research often focuses on protein quantification in model cancer cell lines and cancer tissues. SWATH (sequential windowed acquisition of all theoretical fragment ion spectra), the state of the art method, enables the quantification of all proteins included in spectral library. Spectral library contains fragmentation patterns of each detectable protein in a sample. Thorough spectral library preparation will improve quantitation of low abundant proteins which usually play an important role in cancer. AIM: Our research is focused on the optimization of spectral library preparation aimed at maximizing the number of identified proteins in MCF-7 breast cancer cell line. First, we optimized the sample preparation prior entering the mass spectrometer. We examined the effects of lysis buffer composition, peptide dissolution protocol and the material of sample vial on the number of proteins identified in spectral library. Next, we optimized mass spectrometry (MS) method for spectral library data acquisition. CONCLUSION: Our thorough optimized protocol for spectral library building enabled the identification of 1,653 proteins (FDR < 1%) in 1 µg of MCF-7 lysate. This work contributed to the enhancement of protein coverage in SWATH digital biobanks which enable quantification of arbitrary protein from physically unavailable samples. In future, high quality spectral libraries could play a key role in preparing of patient proteome digital fingerprints.Key words: biomarker - mass spectrometry - proteomics - digital biobanking - SWATH - protein quantificationThis 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: 7. 5. 2016Accepted: 9. 6. 2016.

[PDLIM2 and Its Role in Oncogenesis--Tumor Suppressor or Oncoprote?]. / PDLIM2 a jeho role v onkogenezi--tumor supresor nebo onkoprotein?

PDZ and LIM domain containing protein 2 (PDLIM2), also known as Mystique or SLIM, is a member of the actinin-associated LIM family of proteins that play essential roles in cytoskeletone organization, cell differentiation and have been associated with oncogenesis. PDLIM2 is cytoskeletal and nuclear protein encoded by the Mystique gene localized on chromosome 8p21. PDLIM2 regulates stability and activity of several transcription factors, e. g. NF  κB or STAT, and its deregulation is associated with several malignancies. PDLIM2 expression has been connected with both tumor suppression and tumorigenesis. PDLIM2 levels are epigenetically suppressed in different cancers due to Mystique promoter hypermetylation that blocks its transcription. PDLIM2 re expression is able to inhibit tumorigenicity and induces tumor cell death both in vitro and in vivo, which suggest potential tumor suppressor role of PDLIM2. On the other hand, PDLIM2 is highly expressed in cancer cell lines derived from metastatic cancer and its expression is associated with tumor progression and metastasis formation, indicating pro oncogenic role of PDLIM2. The aim of this review is to summarize current knowledge on the role of PDLIM2 in tumor formation and development, focusing on its prospective role as therapeutic target and offering potential explanations of its different functions in oncogenesis that were identified so far.

[Methods for studying tumor cell migration and invasiveness]. / Metody studia bunecné migrace a invazivity nádorových bunek.

Migration and invasiveness are phenotypic characteristics of cells that contribute to physiological processes, such as wound healing or embryogenesis and they are involved in serious pathological processes, namely in tumor cell metastasis. Availability of methods for studying migration and invasiveness of the cells is important for understanding molecular basis of these processes. In the case of cancer, migration, invasiveness and metastatic potential of tumor cells are key factors that determine clinical prognosis of the patients. This communication provides an overview of in vitro and in vivo methods which are used to study cell migration, invasion and metastasis. In vitro meth-ods for studying cell migration include simple two dimensional assays (scratch -  wound assay and the assay based on the effect of hepatocyte growth factor) and methods based on chemotaxis (Dunns chamber, videomicroscopy of cells, the use of carriers with chemoattractants). Methods for studying both cell migration and invasiveness in vitro include more complex systems based on the principle of the Boyden chamber (transwell migration/ invasive test, analysis of cell migration and invasion in xCELLigence system, confocal microscopy based approaches) as well as analysis of cell migration in microchannels. Our overview of in vivo methods provides an introduction into model organisms and methods used in this field, with an emphasis on the study of cancer metastasis in mouse models. The methods described in this review are mainly involved in larger research projects aiming at developing new diagnostic and therapeutic approaches in oncology.

[Techniques to study transendothelial migration in vitro]. / Moznosti studia transendoteliální migrace in vitro.

The most dangerous aspect of cancer is the metastatic spread to other parts of the body. Cancer cells frequently use circulation to spread to secondary locations. By entering the blood-stream (in a process called intravasation) and by crossing the vessel walls at the metastatic sites (extravasation) tumor cells disseminate to distal organs and eventually form life  threatening metastases. Crossing the vessel walls (transendothelial migration) is a vital step of metastatic cascade and the elucidation of mechanisms involved in transendothelial migration might inspire new strategies of targeted antimetastatic therapy. There are several methods to study transendothelial migration in living models (in vivo). Although they offer complex physiological microenvironment, they are expensive and technically demanding, therefore not widely used. As an alternative, sophisticated techniques to investigate transendothelial migration in vitro have been developed. They are generally more available and feasible, but there is still considerable variability in the difficulty of performance, the requirements for specialized devices, accuracy of in vivo simulation and relevance for oncological applications. The classification, various modifications, pros and cons of in vitro techniques for studying transendothelial migration are summarized in this review.

[p SRM, SWATH and HRM -  targeted proteomics approaches on TripleTOF 5600+ mass spectrometer and their applications in oncology research]. / p SRM, SWATH a HRM -  cílené proteomické prístupy na hmotnostním spektrometru TripleTOF 5600+ a jejich aplikace v onkologickém výzkumu.

Development of novel diagnostic and therapeutic approaches in cancer research requires sensitive and quantitative assays for determination of cancer associated proteins in clinical samples. Novel quantitative targeted proteomic approaches are overviewed in this communication. A major advantage of selected reaction monitoring (SRM) and pseudo- SRM lies in the selective and sensitive quantification of selected proteins in large sample sets. As such, they represent an alternative to immunochemical approaches. On the other hand, the potential of HRM and SWATH lies in recording of digital fingerprints, which enable post acquisition quantitative proteomic data mining on a similar basis to SRM. This article shows applications of targeted proteomics in a number of cancer research studies where they were used for quantification and validation of current or potential protein bio-markers and to study their role in cancer development and progression.

Identification and characterisation of pro-metastatic targets, pathways and molecular complexes using a toolbox of proteomic technologies.

BACKGROUND: Cancer metastasis involves changes in signalling pathways, cell adhesion, migration and invasiveness. Modern proteomic, mass spectrometry based techniques enable discovery of new pro-metastatic proteins and their functional partners. Also, they might be involved in their functional characterisation and validation towards development of new diagnostic and therapeutic approaches. AIM: The aim of this communication is to describe current possibilities for proteomic techniques in the discovery and characterization of pro-metastatic targets. The NF-kappaB pathway is one of the players responsible for a number of pro-metastatic processes. The related proteins can be discovered using untargeted proteomic approaches by comparing proteomes with different metastatic potential. Stable isotope labelling based methods enable a parallel analysis of more tumour samples. The identified pro-metastatic proteins can be characterised in relationship to cell migration, invasiveness and proliferation and in terms of their involvement in molecular complexes via protein-protein interactions. Advantages of the metabolic labelling based methods can be taken in these studies, the same applies for characterisation of related surface proteins involved in cell adhesion, invasiveness and cell-to-cell communication. For clinical validation of pro-metastatic proteins in large sample cohorts, approaches of targeted proteomics based on selected reaction monitoring are becoming methods of choice. CONCLUSION: Current proteomics methods play an important role in the identification of novel pro-metastatic proteins, pathways and molecular complexes, in their functional characterisation and validation towards diagnostic and therapeutic application.

Fe/Mn superoxide dismutase-encoding gene in Paracoccus denitrificans is induced by azide and expressed independently of the FNR-type regulators.

Paracoccus denitrificans cells undergo changes in protein composition upon exposure to azide, a known activator of the fumarate-nitrate reduction (FNR)-type transcription factor NarR. One of the most prominent protein species inducible by azide is a Fe/Mn-family superoxide dismutase (SOD). Azide induces SOD at protein, mRNA transcript, and enzyme activity levels in the aerobically growing cells. Since SOD expression remains unaffected in the fnrP-, nnr-, and narR-mutant strains, we postulate a mechanism independent of the known FNR-type regulators but involving a redox signal arising from the respiratory chain.

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.

Application of capillary zone electrophoresis to study the properties of rhodanese from Acidithiobacillus ferrooxidans.

A new capillary zone electrophoretic method was applied to the assay of enzymic activity of rhodanese from Acidithiobacillus ferroxidans. The enzyme activity determined by capillary zone electrophoresis was compared with that determined by discontinuous spectrophotometry, the values obtained being in good agreement. The method was also used to evaluate Michaelis constants of cyanide and thiocyanate as substrates; a new approach was developed to solve the problem with variable ionic strength of the samples. The pH and temperature optima for the enzyme were also determined.

Determination of rhodanese enzyme activity by capillary zone electrophoresis.

A new sensitive method has been developed for the determination of rhodanese activity. The enzymatic reactions were carried out directly in thermostatted autosampler vials and the formation of SCN- was monitored by sequential capillary zone electrophoretic runs. The determinations were performed in a 75-micron fused-silica capillary using 0.1 M beta-alanine-HCl (pH 3.50) as a background electrolyte, a separation voltage of 18 kV (negative polarity), a capillary temperature of 25 degrees C and direct detection at 200 nm. Short-end injection or long-end injection procedures were used for sample application. The method is rapid, able to be automated and requires only small amounts of sample and substrates, which is especially important in the case of highly toxic cyanide. The developed capillary electrophoretic method also has great potential for thiocyanate determinations in other applications.