Východiska: Glykosylace jsou posttranslační modifikace zodpovědné za mnohé buněčné procesy vč. interakcí mezi proteiny, za buněčnou signalizaci nebo regulaci buněčného cyklu. Změny glykosylací na sérových proteinech reflektují stav buněk a tkání v organizmu a ukazují se proto jako užitečný marker pro diagnostiku přítomnosti a progrese nádorových onemocnění a jejich subtypizaci. Pro charakterizaci změn je hojně využíváno tzv. glykanové profilování, tedy měření N-glykanů uvolněných ze sérových proteinů. S rozvojem analytické instrumentace a pokročilých bioinformatických postupů se pro charakterizaci N-glykosylací prosazuje stále více glykoproteomický přístup, který vedle glykomické informace zachovává i informaci proteomickou, tj. znalost původního proteinu a glykosylačního místa. Cíl: Cílem této přehledové práce je zmapovat dosavadní studie popisující změny glykosylací na sérových proteinech během nádorových onemocnění. Pozornost je věnována glykomickým i glykoproteomickým přístupům. Poslední část článku shrnuje analytické metody používané pro tento typ analýz.

Background: Glycosylation is a posttranslational modification responsible for many biological 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 diagnosis 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.

• Klíčová slova
• glykoproteomika, N-glykanové profilování,
• MeSH
• glykomika * MeSH
• glykopeptidy analýza   MeSH
• hmotnostní spektrometrie metody   MeSH
• klinická studie jako téma MeSH
• krevní proteiny analýza   MeSH
• lidé MeSH
• metaanalýza jako téma MeSH
• nádory * patofyziologie   MeSH
• polysacharidy analýza   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• přehledy MeSH

We firstly identified 48 kDa molecular form of the unconventional myosin 1c (p48/Myo1C), and isolated it from blood serum of multiple sclerosis patients. The amount of p48/Myo1C in human blood serum correlated with some autoimmune, hemato-oncological and neurodegenerative diseases and thus may serve as a potential molecular biomarker. The biological functions of this protein in human blood remain unknown. Previously, we used the monodisperse magnetic poly (glycidyl methacrylate)(mag-PGMA-NH2 ) microspheres with immobilized 48/Myo1C and western-blot analysis, which allowed us to identify IgM and IgG immunoglobulins presenting an affinity to this protein. Here, we used mass spectrometry followed by the western blotting in order to identify other blood serum proteins with affinity to 48/Myo1C. The obtained data demonstrate that 48/Myo1C binds to component 3 of the complement and the antithrombin-III proteins. A combination of magnetic microparticle-based affinity chromatography with MALDI-TOF mass spectrometry and an in silico analysis provided an opportunity to identify the partners of interaction of 48/Myo1C with other proteins, in particular those participating in complement and coagulation cascades.

• MeSH
• chromatografie afinitní metody   MeSH
• krevní proteiny analýza   chemie   metabolismus   MeSH
• lidé MeSH
• magnety MeSH
• mikrosféry MeSH
• molekulární modely MeSH
• myosin typu I chemie   metabolismus   MeSH
• prognóza MeSH
• roztroušená skleróza krev   MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice metody   MeSH
• vazba proteinů MeSH
• western blotting MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

At present, Alzheimer's disease is detected mainly using psychological tests, which can only confirm the disease in its more advanced phases. Therefore, bioanalytical possibilities for detecting this disease earlier are being investigated. To date, the results of analyses, which focus mainly on the study of lipids and proteins either in cerebrospinal fluid or much less often in blood plasma, do not provide satisfactory results. In addition, cerebrospinal fluid sampling is uncomfortable for the patients and involves many health risks. In this work, we deal with proteomic analysis using Matrix-Assisted Laser Desorption/Ionisation-Time of Flight and Liquid Chromatography coupled to tandem Mass Spectrometry of blood plasma with a focus on various ways of preanalytical sample treatments. This should lead to results improvement and facilitate the subsequent evaluation using principal component analysis and partial least squares discriminant analysis. The obtained results indicate the direction of further research, namely the study of interactions between proteins and lipids contained in blood plasma. These substances may be regarded as potential biomarkers allowing for the diagnosis of Alzheimer ́s disease even in its early stages.

• MeSH
• Alzheimerova nemoc * krev   diagnóza   MeSH
• biologické markery krev   MeSH
• chromatografie kapalinová metody   MeSH
• krevní plazma chemie   MeSH
• krevní proteiny analýza   MeSH
• lidé středního věku MeSH
• lidé MeSH
• lipidy krev   MeSH
• metabolismus lipidů MeSH
• proteomika metody   MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice metody   MeSH
• tandemová hmotnostní spektrometrie metody   MeSH
• Check Tag
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH

INTRODUCTION: Thyroid cytology is a widely accepted tool in the clinical triaging of nodular lesions. Cell blocks (CBs) can help in the diagnosis of atypical lesions, namely, thyroid Bethesda category of Atypia of undetermined significance/follicular lesion of undetermined significance (AUS/FLUS). METHODS: In a series of 224 AUS/FLUS thyroid samples with CB, we studied CB cellularity and feasibility of 3 immunohistochemical markers (cytokeratin 19 [CK19], HBME-1, and galectin-3) apart and in combination. RESULTS: The CBs were non-diagnostic in 34 cases. Twenty-four CBs contained <10 cells, 45 CBs 10-50 cells, and 121 CBs >50 cells. Notably, more cellularity was found in CBs performed by plasma-thrombin and in-house techniques (p < 0.001). The diagnostic accuracy to detect malignancy was 65.1% for CK19, 72.1% for HBME-1, and 70.3% for galectin-3. CONCLUSION: In conclusion, CB cellularity is essential for successful immunohistochemistry application and further diagnostic workup of AUS/FLUS cases.

• MeSH
• fixace tkání MeSH
• galektiny analýza   MeSH
• imunohistochemie * MeSH
• keratin-19 analýza   MeSH
• krevní proteiny analýza   MeSH
• lidé MeSH
• nádorové biomarkery analýza   MeSH
• nádory štítné žlázy chemie   patologie   MeSH
• prediktivní hodnota testů MeSH
• reprodukovatelnost výsledků MeSH
• retrospektivní studie MeSH
• studie proveditelnosti MeSH
• stupeň nádoru MeSH
• zalévání tkání do parafínu * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• srovnávací studie MeSH

The study aimed to investigate free light chain (FLC) monoclonality in patients with an abnormal free kappa/lambda ratio (FLC ratio). Seventy serum samples with abnormal FLC ratio were examined using an immunoturbidimetry (Binding Site, SPA) and the two different enzyme-linked immunosorbent assays (1. Sebia diagnostic kit; 2. in house methods), the monoclonal or oligoclonal bands of (FLC) by immunofixation electrophoresis (IE) and isoelectric focusing followed by affinity immunoblotting (IEF/AIB). The reference interval was calculated by non-parametric percentile method. 5.7% of samples examined by IE were suspected of monoclonal character of FLCs, but subsequently monoclonality was refuted by more sensitive IEF/AIB method; 7%, resp. 2.9% of samples showed FLC kappa, resp. FLC lambda oligoclonal character of bands. A statistically significant dependence was found between FLC ratio (Sebia) and FLC ratio (SPA), rs = 0.510, p = .001. Kappa statistic evaluated a fair conformity between the FLC ratio (Sebia) and IEF/AIB (kappa = 0.468) and between FLC ratio (in house) and IEF/AIB (kappa = 0.300). The verified reference interval for FLC ratio (Binding Site) is between 0.35 and 2.18. The IEF/AIB is the most sensitive method to discriminate between monoclonal and oligoclonal bands of FLC. The Binding Site and Sebia diagnostic kits do not give consistent results. The Binding Site diagnostic kit provides more results above reference interval of FLC ratios. For routine decision on monoclonality of the FLC ratio (SPA) it is advisable to use a verified reference interval.

• MeSH
• dospělí MeSH
• imunoglobuliny - lambda-řetězce analýza   imunologie   MeSH
• isoelektrická fokusace metody   MeSH
• krevní proteiny analýza   MeSH
• lidé středního věku MeSH
• lidé MeSH
• monoklonální protilátky analýza   imunologie   MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• srovnávací studie MeSH

N-Acetyllactosamine (LacNAc; Galβ4GlcNAc) is a typical disaccharide ligand of galectins. The most abundant members of these human lectins, galectin-1 (Gal-1) and galectin-3 (Gal-3), participate in a number of pathologies including cancerogenesis and metastatic formation. In this study, we synthesized a series of fifteen N-(2-hydroxypropyl)methacrylamide (HPMA)-based glycopolymers with varying LacNAc amounts and presentations and evaluated the impact of their architecture on the binding affinity to Gal-1 and Gal-3. The controlled radical reversible addition-fragmentation chain transfer copolymerization technique afforded linear polymer precursors with comparable molecular weight (Mn ≈ 22,000 g mol-1) and narrow dispersity (D̵ ≈ 1.1). The precursors were conjugated with the functionalized LacNAc disaccharide (4-22 mol % content in glycopolymer) prepared by enzymatic synthesis under catalysis by β-galactosidase from Bacillus circulans. The structure-affinity relationship study based on the enzyme-linked immunosorbent assay revealed that the type of LacNAc presentation, individual or clustered on bi- or trivalent linkers, brings a clear discrimination (almost 300-fold) between Gal-1 and Gal-3, reaching avidity to Gal-1 in the nanomolar range. Whereas Gal-1 strongly preferred a dense presentation of individually distributed LacNAc epitopes, Gal-3 preferred a clustered LacNAc presentation. Such a strong galectin preference based just on the structure of a multivalent glycopolymer type is exceptional. The prepared nontoxic, nonimmunogenic, and biocompatible glycopolymers are prospective for therapeutic applications requiring selectivity for one particular galectin.

• MeSH
• akrylamidy chemie   MeSH
• aminocukry chemie   MeSH
• Bacillus enzymologie   MeSH
• beta-galaktosidasa metabolismus   MeSH
• disacharidy chemická syntéza   MeSH
• ELISA MeSH
• epitopy MeSH
• galektin 1 analýza   metabolismus   MeSH
• galektiny analýza   metabolismus   MeSH
• katalýza MeSH
• krevní proteiny analýza   metabolismus   MeSH
• magnetická rezonanční spektroskopie MeSH
• polymerizace MeSH
• polymery chemie   metabolismus   farmakologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The steadily increasing incidence of malignant melanoma (MM) and its aggressive behaviour makes this tumour an attractive cancer research topic. The tumour microenvironment is being increasingly recognised as a key factor in cancer biology, with an impact on proliferation, invasion, angiogenesis and metastatic spread, as well as acquired therapy resistance. Multiple bioactive molecules playing cooperative roles promote the chronic inflammatory milieu in tumours, making inflammation a hallmark of cancer. This specific inflammatory setting is evident in the affected tissue. However, certain mediators can leak into the systemic circulation and affect the whole organism. The present study analysed the complex inflammatory response in the sera of patients with MM of various stages. Multiplexed proteomic analysis (Luminex Corporation) of 31 serum proteins was employed. These targets were observed in immunohistochemical profiles of primary tumours from the same patients. Furthermore, these proteins were analysed in MM cell lines and the principal cell population of the melanoma microenvironment, cancer‑associated fibroblasts. Growth factors such as hepatocyte growth factor, granulocyte‑colony stimulating factor and vascular endothelial growth factor, chemokines RANTES and interleukin (IL)‑8, and cytokines IL‑6, interferon‑α and IL‑1 receptor antagonist significantly differed in these patients compared with the healthy controls. Taken together, the results presented here depict the inflammatory landscape that is altered in melanoma patients, and highlight potentially relevant targets for therapy improvement.

• MeSH
• chemokiny krev   MeSH
• dospělí MeSH
• fibroblasty asociované s nádorem metabolismus   MeSH
• krevní proteiny analýza   MeSH
• lidé středního věku MeSH
• lidé MeSH
• melanom krev   metabolismus   MeSH
• nádorové biomarkery krev   MeSH
• nádorové buněčné linie MeSH
• pilotní projekty MeSH
• prognóza MeSH
• proteomika metody   MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• studie případů a kontrol MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• srovnávací studie MeSH

The cytopoxic effect of RL2 lactaptin (the recombinant analog of proteolytic fragment of human kappa-casein) toward tumor cells in vitro and in vivo presents it as a novel promising antitumor drug. The binding of any drug with serum proteins can affect their activity, distribution, rate of excretion and toxicity in the human body. Here, we studied the ability of RL2 to bind to various blood serum proteins. Using magnetic microparticles bearing by RL2 as an affinity matrix, in combination with mass spectrometry and western blot analysis, we found a number of blood serum proteins possessing affinity for RL2. Among them IgA, IgM and IgG subclasses of immunoglobulins, apolipoprotein A1 and various cortactin isoforms were identified. This data suggests that in the bloodstream RL2 lactaptin takes part in complicate protein-protein interactions, which can affect its activity.

• MeSH
• antitumorózní látky metabolismus   MeSH
• chromatografie afinitní metody   MeSH
• kaseiny metabolismus   MeSH
• krevní proteiny analýza   izolace a purifikace   metabolismus   MeSH
• kyseliny polymethakrylové chemie   MeSH
• lidé MeSH
• magnety chemie   MeSH
• mikrosféry MeSH
• rekombinantní proteiny metabolismus   MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

The study aim was to compare molecular-level effects (blood-dialyzer interactions) of heparin and citrate anticoagulation using proteome-wide analysis of biofilm adsorbed to dialysis membrane. Ten patients receiving maintenance hemodialysis were examined in a crossover design under three different anticoagulation regimens, namely citrate, heparin, and anticoagulation-free (control). Following a regular hemodialysis session (4 hours, polysulfone membrane), dialyzers were flushed and the surface biofilm eluted by acetic acid. Protein composition of the eluates was determined by 2-dimensional gel electrophoresis and resulting patterns compared between regimens. Proteins responsible for the difference were identified by mass spectrometry. Citrate anticoagulation was associated with significantly less protein adsorption to the membrane than heparin (2.2 [1.1-2.9] mg vs. 6.5 [2.9-11.6] mg, P = 0.009). Among the proteins identified as major discriminators between citrate and the other regimens, fibrin α-chain fragments of molecular weight below 40 kDa prevailed. In these fragments, an analysis of the amino acid sequence has been performed by comparison with the UniProt database. It showed missing α-chain cross-links. On the contrary, heparin prevented adsorption and cleavage of several heparin-binding proteins; especially complement factor H-related protein 3, insulin-like growth factor binding proteins (2, 4, and 5), and chemerin. Compared to heparin, citrate is associated with less protein adsorption and imperfectly crosslinked fibrin clot formation. Membrane adsorptive properties are significantly modified by the anticoagulation regimen.

• MeSH
• adsorpce MeSH
• antikoagulancia farmakologie   MeSH
• dialýza ledvin přístrojové vybavení   metody   MeSH
• hemokoagulace účinky léků   MeSH
• heparin farmakologie   MeSH
• krevní proteiny analýza   MeSH
• kyselina citronová farmakologie   MeSH
• lidé středního věku MeSH
• lidé MeSH
• membrány umělé MeSH
• polymery chemie   MeSH
• proteomika MeSH
• senioři MeSH
• sulfony chemie   MeSH
• Check Tag
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH

OBJECTIVES: With over 35 million cases worldwide, Alzheimer's disease (AD) represents the main cause of dementia. The differentiation of AD from other types of dementia is challenging and its early diagnosis is complicated. The established biomarkers are not only based on the invasive collection of cerebrospinal fluid, but also lack sufficient sensitivity and specificity. Therefore, much current effort is aimed at the identification of new biomarkers of AD in peripheral blood. DESIGN AND METHODS: We focused on blood-based analyses using chiroptical spectroscopy (Raman optical activity, electronic circular dichroism) supplemented with conventional vibrational spectroscopy (infrared, Raman) and metabolomics (high-performance liquid chromatography with a high-resolution mass detection). RESULTS: This unique approach enabled us to identify the spectral pattern of AD and variations in metabolite levels. Subsequent linear discriminant analysis of the spectral data resulted in differentiation between the AD patients and control subjects. CONCLUSIONS: It may be stated that this less invasive approach has strong potential for the identification of disease-related changes within essential plasmatic biomolecules and metabolites.

• MeSH
• Alzheimerova nemoc krev   diagnóza   MeSH
• biologické markery krev   MeSH
• cirkulární dichroismus MeSH
• diskriminační analýza MeSH
• krevní proteiny analýza   MeSH
• lidé středního věku MeSH
• lidé MeSH
• metabolomika MeSH
• Ramanova spektroskopie MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• Check Tag
• lidé středního věku MeSH
• lidé MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• Publikační typ
• časopisecké články MeSH