The use of contaminated raw materials can lead to the transfer of mycotoxins into the final product, including beer. This study describes the use of the commercially available immunoaffinity column 11+Myco MS-PREP® and UPLC-MS/MS for the determination of mycotoxins in pale lager-type beers brewed in Czech Republic and other European countries. The additional aim of the work was to develop, optimize and validate this analytical method. Validation parameters such as linearity, limit of detection (LOD), limit of quantification (LOQ), precision and accuracy were tested. The calibration curves were linear with correlation coefficients (R2 > 0.99) for all mycotoxins under investigation. The LOD ranged from 0.1 to 50 ng/L and LOQ from 0.4 to 167 ng/L. Recoveries of the selected analytes ranged from 72.2 to 101.1%, and the relative standard deviation under conditions repeatability (RSDr) did not exceed 16.3% for any mycotoxin. The validated procedure was successfully applied for the analysis of mycotoxins in a total of 89 beers from the retail network. The results were also processed using advanced chemometric techniques and compared with similar published studies. The toxicological impact was taken into account.

• MeSH
• chromatografie kapalinová metody   MeSH
• dietární expozice analýza   MeSH
• mykotoxiny * analýza   MeSH
• pivo analýza   MeSH
• tandemová hmotnostní spektrometrie metody   MeSH
• vysokoúčinná kapalinová chromatografie metody   MeSH
• Publikační typ
• časopisecké články MeSH

BACKGROUND: Sepsis is a common worldwide health condition with high mortality. It is caused by a dysregulated immune response to the pathogen. Severe infections resulting in sepsis can be also determined by monitoring several bloodstream biomarkers, one of them being pro-hormone procalcitonin (PCT). PCT concentration in the bloodstream correlates well with sepsis and in severe cases increases up to a thousand times from the healthy physiological values in a short time. In this study, we developed a rapid technique for PCT detection by MALDI-TOF mass spectrometry, that uses in-situ enrichment directly on the specialized immuno MALDI chips that are utilized as MALDI plates. The method's ability to detect PCT was confirmed by comparing the results with LC-MS bottom-up workflow. The new method detects intact PCT by its m/z and uncovers its alternations in septic serum. METHODS: The MALDI chips used for the detection of PCT were prepared by ambient ion soft landing of anti-PCT antibody on an ITO glass slide. The chips were used for the development of the rapid MALDI-TOF MS method. A parallel method based on affinity enrichment on magnetic beads followed by LC-MS/MS data-dependent peptide microsequencing was used to prove PCT presence in the sample. All samples were also tested by ELISA to determine PCT concentration prior to analyzing them by mass spectrometry methods. RESULTS: The MALDI chip method was optimized using recombinant PCT spiked into the human serum. The PCT detection limit was 10 ng/mL. The optimized method was used to analyze 13 sera from patients suffering sepsis. The PCT results were confirmed by LC-MS/MS. The measurement of the intact PCT by the MALDI chip method revealed that sera of patients with severe sepsis have other forms of PCT present, which show post-processing of the primary sequence by cleavage of PCT, resulting in the formation of N and C termini fragments. CONCLUSIONS: Procalcitonin from human serum was successfully enriched and detected using immunoaffinity MALDI chips. The intact PCT was characterized in 13 septic patients. The method is more specific compared to non-MS-based immunoaffinity techniques and allows observation of different variants of PCT in septic patients.

• Publikační typ
• časopisecké články MeSH

INTRODUCTION: N-glycosylation is a ubiquitous and variable posttranslational modification that regulates physiological functions of secretory and membrane-associated proteins and the dysregulation of glycosylation pathways is often associated with cancer growth and metastasis. Prostate-specific membrane antigen (PSMA) is an established biomarker for prostate cancer imaging and therapy. METHODS: Mass spectrometry was used to analyze the distribution of the site-specific glycoforms of PSMA in insect, human embryonic kidney, and prostate cancer cells, and in prostate tissue upon immunoaffinity enrichment. RESULTS: While recombinant PSMA expressed in insect cells was decorated mainly by paucimannose and high mannose glycans, complex, hybrid, and high mannose glycans were detected in samples from human cells and tissue. We noted an interesting spatial distribution of the glycoforms on the PSMA surface-high mannose glycans were the dominant glycoforms at the N459, N476, and N638 sequons facing the plasma membrane, while the N121, N195, and N336 sites, located at the exposed apical PSMA domain, carried primarily complex glycans. The presence of high mannose glycoforms at the former sequons likely results from the limited access of enzymes of the glycosynthetic pathway required for the synthesis of the complex structures. In line with the limited accessibility of membrane-proximal sites, no glycosylation was observed at the N51 site positioned closest to the membrane. CONCLUSIONS: Our study presents initial descriptive analysis of the glycoforms of PSMA observed in cell lines and in prostate tissue. It will hopefully stimulate further research into PSMA glycoforms in the context of tumor staging, noninvasive detection of prostate tumors, and the impact of glycoforms on physicochemical and enzymatic characteristics of PSMA in a tissue-specific manner.

• MeSH
• antigeny povrchové metabolismus   MeSH
• buněčné linie MeSH
• glutamátkarboxypeptidasa II metabolismus   MeSH
• glykosylace MeSH
• hmotnostní spektrometrie metody   MeSH
• lidé MeSH
• nádorové biomarkery analýza   MeSH
• nádory prostaty * metabolismus   patologie   MeSH
• polysacharidy * klasifikace   metabolismus   MeSH
• posttranslační úpravy proteinů MeSH
• prostata * enzymologie   metabolismus   patologie   MeSH
• staging nádorů MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

Blood sausages consisting of groats, pork, porcine offal, fat, blood, and spices are very popular in the Czech Republic. All these ingredients are potential sources of dietary exposure to ochratoxin A (OTA). OTA has a strong affinity to serum proteins in porcine blood. Thus, the contamination of blood sausages with OTA can be expected. This study aims to evaluate OTA in 200 samples of porcine blood sausages purchased at the Czech market during 2020-2021. The analytical method high-performance liquid chromatography coupled with fluorescence detection with pre-treatment using immunoaffinity columns was employed to determine OTA. The limit of detection was 0.03 ng/g and the limit of quantification 0.10 ng/g. Recovery was 71.6 %. All samples were positive at contents ranging from 0.15 to 5.68 ng/g with a mean of 1.47 ng/g, and a median of 1.26 ng/g. A total of 66% of these samples contained OTA content exceeding the maximum limit of 1 ng/g set in Italy. This study demonstrates that the Czech population is exposed to OTA from blood sausages. The proposed preliminary action limit for OTA in blood sausages should be set at 1 ng/g. No regulatory limits for OTA in blood sausages have been established yet in the European Union legislation. To protect human health, further monitoring of OTA in these products is necessary.

• MeSH
• kontaminace potravin analýza   MeSH
• masné výrobky * analýza   MeSH
• ochratoxiny * analýza   MeSH
• prasata MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Česká republika MeSH

Ochratoxin A (OTA) exposure can result in chronic renal diseases and cancer. The incidence of kidney, renal pelvis, and ureter malignant neoplasms in the Czech Republic is approximately 29.5 renal tumours per 100,000 inhabitants. The question arises whether mycotoxins are also involved in kidney disease and cancer. A sensitive validated analytical methodology, based on an immunoaffinity clean-up followed by HPLC with fluorescence detection, was developed to explore whether OTA accumulates in clear renal cell carcinoma-adenocarcinoma in Czech patients. Simultaneously, DNA-adducts and OTA metabolites were qualitatively analysed in tissues and urine. OTA was analysed in 33 kidney and tumour samples from 26 men and 7 women collected during nephrectomy from patients of the East Bohemian region from 2015 to 2017. OTA was found in 76% of the analysed samples. Its concentrations ranged from not detectable to 390 ng/kg with a median of 167 ng/kg in kidney samples and from not detectable to 430 ng/kg with a median of 122 ng/kg in tumour samples. Urinary OTA metabolites and DNA adducts were qualitatively analysed for the corresponding 20 patients. The presence of some OTA metabolites such as ochratoxin A hydroquinone and/or decarboxylated ochratoxin A hydroquinone correlate with the presence of OTA-DNA adducts.

• MeSH
• adukty DNA MeSH
• karcinom z renálních buněk * metabolismus   patologie   MeSH
• ledviny metabolismus   MeSH
• lidé středního věku MeSH
• lidé MeSH
• nádorové biomarkery analýza   MeSH
• nádory ledvin * metabolismus   patologie   MeSH
• ochratoxiny analýza   MeSH
• senioři MeSH
• Check Tag
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH

An aberrant immune response developed early in life may trigger inflammatory bowel disease (IBD) and food allergies (e.g., celiac disease). Fecal levels of immune markers categorize an inflammatory response (e.g., food allergy, autoimmune) paralleled with the initial microbial colonization. The immunoaffinity assays are routinely applied to quantify circulating immune protein markers in blood/serum. However, a reliable, multiplex assay to quantify fecal levels of immune proteins is unavailable. We developed mass spectrometry assays to simultaneously quantify fecal calprotectin, myeloperoxidase, eosinophil-derived neurotoxin, eosinophil cationic protein, alpha-1-antitrypsin 1, and adaptive immunity effectors in 134 neonatal stool swabs. We optimized extraction and proteolytic protocol and validated the multiplex assay in terms of linearity of response (> 100; typically 0.04 to 14.77 µg/mg of total protein), coefficient of determination (R2; > 0.99), the limit of detection (LOD; 0.003 to 0.04 µg/mg of total protein), the limit of quantification (LOQ; 0.009 to 0.122 µg/mg of total protein) and robustness. The median CV of intra- and interday precision was 9.8% and 14.1%, respectively. We quantified breast milk-derived IGHA2 to differentiate meconium from feces samples and to detect the first food intake. An early life profiling of immune markers reflects disrupted intestinal homeostasis, and it is perhaps suitable for pre-symptomatic interception of IBD and food allergies.

• MeSH
• biologické markery metabolismus   MeSH
• chemické techniky analytické metody   MeSH
• feces chemie   MeSH
• hmotnostní spektrometrie metody   MeSH
• idiopatické střevní záněty etiologie   metabolismus   MeSH
• lidé MeSH
• novorozenec MeSH
• odběr biologického vzorku metody   MeSH
• potravinová alergie etiologie   metabolismus   MeSH
• zánět diagnóza   mikrobiologie   MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• novorozenec MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The Czech Republic occupies the first place in the world in the frequency of renal and other urinary tract tumours, but their aetiology is unknown. To explore whether carcinogenic and nephrotoxic mycotoxins may contribute to kidney diseases in the Czech population, biomarkers of ochratoxin A (OTA) and citrinin (CIT) exposure were determined in biological specimens from a cohort of 50 patients with malignant renal tumours. Biomarker analyses in blood and urine samples used validated targeted methods for measuring OTA and CIT plus dihydrocitrinone (DH-CIT) after enrichment of analytes by specific immunoaffinity clean-up. OTA and CIT plus its metabolite DH-CIT were frequently detected in patient urine samples (OTA 62%; CIT 91%; DH-CIT 100%). The concentration ranges in urine were 1-27.8 ng/L for OTA, 2-87 ng/L for CIT and 2-160 ng/L for DH-CIT. The analyses of blood samples revealed also a frequent co-occurrence of OTA and CIT, in the ranges of 40-870 ng/L serum for OTA and 21-182 ng/L plasma for CIT. This first analysis of biomarkers in blood and urine samples of Czech patients revealed no major differences in comparison with published data for the general healthy Czech and European populations. Nonetheless, a frequent co-occurrence of CIT and OTA biomarkers in patient samples may be of interest with regard to potential interactions with other risk factors for renal disease.

• MeSH
• biologické markery krev   moč   MeSH
• chromatografie kapalinová MeSH
• citrinin krev   moč   MeSH
• dospělí MeSH
• kohortové studie MeSH
• lidé středního věku MeSH
• lidé MeSH
• mykotoxiny krev   moč   MeSH
• nádory ledvin chemie   moč   MeSH
• ochratoxiny krev   moč   MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• tandemová hmotnostní spektrometrie 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
• Geografické názvy
• Československo MeSH

Liquid chromatography (LC) coupled with mass spectrometry (MS) is widely used for the determination of mycotoxins in cereals and cereal-based products. In addition to the regulated mycotoxins, for which official control is required, LC-MS is often used for the screening of a large range of mycotoxins and/or for the identification and characterization of novel metabolites. This review provides insight into the LC-MS methods used for the determination of co-occurring mycotoxins with special emphasis on multiple-analyte applications. The first part of the review is focused on targeted LC-MS approaches using cleanup methods such as solid-phase extraction and immunoaffinity chromatography, as well as on methods based on minimum cleanup (quick, easy, cheap, effective, rugged, and safe; QuEChERS) and dilute and shoot. The second part of the review deals with the untargeted determination of mycotoxins by LC coupled with high-resolution MS, which includes also metabolomics techniques to study the fate of mycotoxins in plants.

• MeSH
• analýza potravin metody   MeSH
• chromatografie afinitní metody   MeSH
• extrakce na pevné fázi metody   MeSH
• houby izolace a purifikace   metabolismus   MeSH
• jedlá semena chemie   metabolismus   mikrobiologie   MeSH
• metabolomika metody   MeSH
• mykotoxiny analýza   metabolismus   MeSH
• tandemová hmotnostní spektrometrie metody   MeSH
• vysokoúčinná kapalinová chromatografie metody   MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

In mass cytometry, the isolation of pure lymphocytes is very important to obtain reproducible results and to shorten the time spent on data acquisition. To prepare highly purified cell suspensions of peripheral blood lymphocytes for further analysis on mass cytometer, we used the new CD81+ immune affinity chromatography cell isolation approach. Using 21 metal conjugated antibodies in a single tube we were able to identify all basic cell subsets and compare their relative abundance in final products obtained by density gradient (Ficoll-Paque) and immune affinity chromatography (CD81+ T-catch™) isolation approach. We show that T-catch isolation approach results in purer final product than Ficoll-Paque (P values 0.0156), with fewer platelets bound to target cells. As a result acquisition time of 105 nucleated cells was 3.5 shorter. We then applied unsupervised high dimensional analysis viSNE algorithm to compare the two isolation protocols, which allowed us to evaluate the contribution of unsupervised analysis over supervised manual gating. ViSNE algorithm effectively characterized almost all supervised cell subsets. Moreover, viSNE uncovered previously overseen cell subsets and showed inaccuracies in Maxpar™ Human peripheral blood phenotyping panel kit recommended gating strategy. These findings emphasize the use of unsupervised analysis tools in parallel with conventional gating strategy to mine the complete information from a set of samples. They also stress the importance of the impurity removal to sensitively detect rare cell populations in unsupervised analysis. © 2016 International Society for Advancement of Cytometry.

• MeSH
• antigeny CD81 chemie   metabolismus   MeSH
• Ficoll chemie   MeSH
• leukocyty mononukleární cytologie   MeSH
• lidé MeSH
• lymfocyty cytologie   imunologie   MeSH
• obrazová cytometrie metody   MeSH
• protilátky chemie   imunologie   MeSH
• separace buněk metody   MeSH
• viabilita buněk imunologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Brassinosteroids (BRs) are plant-specific steroid hormones that play essential roles in the regulation of many important physiological processes in plant life. Their extremely low concentrations (~pmoles/g FW) in plant tissue and huge differences in polarity of individual members within the BR family hamper their detection and quantification. To address this problem, an immunoaffinity sorbent with broad specificity and high capacity for different BR metabolites containing a monoclonal antibody (mAb) against a BR spacer (20S)-2α,3α-dihydroxy-7-oxa-7α-homo-5α-pregnane-6-one-20 carboxylic acid (BR4812) was used for the rapid and highly selective isolation of endogenous BRs containing a 2α,3α-diol in ring A from minute plant samples. This enrichment procedure was successfully applied as a sample preparation method prior to quantitative analysis of BRs in real plant tissues by ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Use of immunoaffinity chromatography (IAC) increased the sensitivity of the UHPLC-MS/MS analysis owing to improvements in the BR signal-to-noise ratio (S/N) and matrix factor (MF). Although MF values of BRs analyzed in classical samples ranged from 8.9% to 47.4%, MF values for the IAC purified samples reached 44.5-96.6%. Thus, the developed IAC-UHPLC-MS/MS approach was shown to be a simple, robust, effective and extremely fast procedure requiring minute amounts of plant samples suitable for the quantitative profiling of many BR metabolites, helping to overcome the major problems associated with their determination in very complex plant matrices.

• MeSH
• Brassica napus chemie   MeSH
• brassinosteroidy analýza   izolace a purifikace   MeSH
• chromatografie afinitní metody   MeSH
• imobilizační protilátky chemie   MeSH
• imunosorbenty chemie   MeSH
• regulátory růstu rostlin analýza   izolace a purifikace   MeSH
• rostlinné extrakty chemie   MeSH
• tandemová hmotnostní spektrometrie metody   MeSH
• vysokoúčinná kapalinová chromatografie metody   MeSH
• Publikační typ
• časopisecké články MeSH