nano-LC–MS Dotaz Zobrazit nápovědu
Although the proteome of each organism is unambiguously coded in its genome, the proteome shows the real biology in action in each particular organism. New powerful tools are being developed for biochemists and biologists to analyze complex biological samples for studying the complete protein supplement of the genome, i. e., the proteome. There are several methods available for proteome analysis including 2-DE and several forms of MS. In recent years, technologies such as microfluidics and array-based systems have appeared in the field of analysis, identification, and quantification of proteins. These novel approaches might help in solving current technical challenges in proteomics. This paper presents a practical application of the first commercially available microfluidic nano-ESI device coupled with nano-LC (i. e., HPLC-chip) for the analysis of samples of some biological protein mixtures.
- MeSH
- chromatografie kapalinová metody přístrojové vybavení MeSH
- čočka chemie MeSH
- financování organizované MeSH
- hmotnostní spektrometrie metody přístrojové vybavení MeSH
- lidé MeSH
- mikrofluidní analytické techniky MeSH
- molekulární sekvence - údaje MeSH
- proteiny analýza genetika MeSH
- rýže (rod) chemie MeSH
- semena rostlinná chemie MeSH
- testování materiálů MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- hodnotící studie MeSH
The history of liquid chromatography started more than a century ago and miniaturization and automation are two leading trends in this field. Nanocolumn liquid chromatography (nano LC) and largely synonymous capillary liquid chromatography (capillary LC) are the most recent results of this process where miniaturization of column dimensions and sorbent particle size play crucial role. Very interesting results achieved in the research of extremely miniaturized LC columns at the end of the last century lacked distinctive raison d'être and only advances in mass spectrometry brought a real breakthrough. Configuration of nano LC-electrospray ionization mass spectrometry (LC-ESI-MS) has become a basic tool in bioanalytical chemistry, especially in proteomics. This review discusses and summarizes past and current trends in the realization of nano liquid chromatography (nano LC) platforms. Special attention is given to the mobile phase delivery under nanoflow rates (isocratic, gradient) and sample injection to the nanocolumn. Available detection techniques applied in nano LC separations are also briefly discussed. We followed up the key themes from the original scientific reports over gradual improvements up to the contemporary commercial solutions.
Cannabis sativa L. is an herbaceous plant belonging to the family of Cannabaceae. It is classified into three different chemotypes based on the different cannabinoids profile. In particular, fiber-type cannabis (hemp) is rich in cannabidiol (CBD) content. In the present work, a rapid nano liquid chromatographic method (nano-LC) was proposed for the determination of the main cannabinoids in Cannabis sativa L. (hemp) inflorescences belonging to different varieties. The nano-LC experiments were carried out in a 100 µm internal diameter capillary column packed with a C18 stationary phase for 15 cm with a mobile phase composed of ACN/H2O/formic acid, 80/19/1% (v/v/v). The reverse-phase nano-LC method allowed the complete separation of four standard cannabinoids in less than 12 min under isocratic elution mode. The nano-LC method coupled to ultraviolet (UV) detection was validated and applied to the quantification of the target analytes in cannabis extracts. The nano-LC system was also coupled to an electrospray ionization-mass spectrometry (ESI-MS) detector to confirm the identity of the cannabinoids present in hemp samples. For the extraction of the cannabinoids, three different approaches, including dynamic maceration (DM), ultrasound-assisted extraction (UAE), and an extraction procedure adapted from the French Pharmacopeia's protocol on medicinal plants, were carried out, and the results achieved were compared.
Desorption electrospray ionization (DESI) mass spectrometry appears to be a useful technique applicable in different areas (e.g. analysis of pharmaceuticals, identification of biologically active compounds in tissues, imaging mass spectrometry). Its modification termed desorption nano-electrospray (nano-DESI) was tested for analysis of anthocyanins. Acidifying of samples and acidic spray liquid (methanol:water=75:25 with 0.2% HCOOH) were essential for obtaining good quality spectra. Profiles of main anthocyanins in wine samples, two vintages (2005 and 2007) of three cultivars (Alibernet, Neronet and Rubinet), were successfully acquired. They were in agreement with results of LC/MS experiments (anthocyanins isolated by solid phase extraction were separated by mu-HPLC with gradient elution and detected by ESI-MS). Nano-DESI-MS data also allowed to determine ratio of two cultivars (Neronet and Rubinet) in their mixture and to detect coloring of wine by tenturier or elderberry extract. Detection of main anthocyanins in slices of wine grape, chokeberries and elderberries or in a wine stain on cotton fabric is also presented.
Polar lipids from the diatoms Diadesmis gallica and Navicula atomus were separated and their structures were determined using high resolution tandem MS HILIC-LC/ESI. This method allowed us to identify 34 classes of lipids, each containing dozens of molecular species, including regioisomers. The largest differences were found in two sulfur-containing lipids, sulfoquinovosyldiacylglycerol and phosphatidylsulfocholine caused probably by the remodeling of lipid species. These diatoms have been found to use several mechanisms to resolve growth in extreme environments, i.e. silica starvation. The presence of insoluble nano-SiO2 leads to the replacement of cellular phospholipids with sulfolipids. Regioisomer ratios also vary depending on the concentration of nano-SiO2 in the culture medium, i.e. the biosynthesis of polar lipids via the prokaryotic (plastidial) and/or eukaryotic (explastidial) pathways. Complex analyses of polar lipids using high resolution HILIC-LC/ESI-tandem, as used for diatoms, can also be used for other photosynthetic microorganisms.
Proteinuria is often used as a surrogate marker in monitoring and predicting outcome in patients with chronic kidney diseases, but it is non-specific. IgAN belongs to the most common primary glomerulonephritis worldwide with serious prognosis. The main aim of this work was to assess differences in urine proteins in patients with IgA nephropathy and to identify abnormal proteins as potential biomarkers of IgA nephropathy or the renal disease. In our pilot project, we selected 20 patients and compared them with 20 healthy volunteers. Protein quantification was performed using iTRAQ (isobaric tag for relative and absolute quantitation) labeling method. The peptides were separated by the isoelectric focusing method (IEF) and nano-LC with C18 column and identified by mass spectrometry using MALDI-TOF/TOF MS. Proteins´ lists obtained from IEF-LC-MS-MS/MS analysis were combined and contained 201 proteins. It was found out that 113 proteins were common in both experiments. 30 urinary proteins were significantly up- or down-regulated in patients with IgA nephropathy. We characterized potential biomarkers such as alpha-1-antitrypsin, apolipoprotein A-I, CD44 antigen or kininogen. Potential biomarkers of IgAN should be validated in further studies.
- MeSH
- alfa-1-antitrypsin genetika moč MeSH
- apolipoprotein A-I genetika moč MeSH
- biologické markery moč MeSH
- dospělí MeSH
- IgA nefropatie diagnóza genetika moč MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladý dospělý MeSH
- pilotní projekty MeSH
- proteomika metody MeSH
- senioři MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladý dospělý MeSH
- mužské pohlaví MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
The bacterial secretome represents a comprehensive catalog of proteins released extracellularly that have multiple important roles in virulence and intercellular communication. This study aimed to characterize the secretome of an environmental isolate Pseudomonas aeruginosa S-8 by analyzing trypsin-digested culture supernatant proteins using nano-LC-MS/MS tool. Using a combined approach of bioinformatics and mass spectrometry, 1088 proteins in the secretome were analyzed by PREDLIPO, SecretomeP 2.0, SignalP 4.1, and PSORTb tool for their subcellular localization and further categorization of secretome proteins according to signal peptides. Using the gene ontology tool, secretome proteins were categorized into different functional categories. KEGG pathway analysis identified the secreted proteins into different metabolic functional pathways. Moreover, our LC-MS/MS data revealed the secretion of various CAZymes into the extracellular milieu, which suggests its strong biotechnological applications to breakdown complex carbohydrate polymers. The identified immunodominant epitopes from the secretome of P. aeruginosa showed the characteristic of being non-allergenic, highly antigenic, nontoxic, and having a low risk of triggering autoimmune responses, which highlights their potential as successful vaccine targets. Overall, the identification of secreted proteins of P. aeruginosa could be important for both diagnostic purposes and the development of an effective candidate vaccine.
- MeSH
- bakteriální proteiny * genetika metabolismus imunologie MeSH
- chromatografie kapalinová MeSH
- kovy metabolismus MeSH
- proteomika MeSH
- Pseudomonas aeruginosa * imunologie metabolismus genetika MeSH
- sekretom * metabolismus imunologie MeSH
- tandemová hmotnostní spektrometrie * MeSH
- výpočetní biologie * MeSH
- Publikační typ
- časopisecké články MeSH
Mucin-type O-glycosylation occurs on many proteins that transit the Golgi apparatus. These glycans impact structure and function of many proteins and have important roles in cellular biosynthetic processes, signaling and differentiation. Although recent technological advances have enhanced our ability to profile glycosylation of glycoproteins, limitations in the understanding of the biosynthesis of these glycan structures remain. Some of these limitations stem from the difficulty to track the biosynthetic process of mucin-type O-glycosylation, especially when glycans occur in dense clusters in repeat regions of proteins, such as the mucins or immunoglobulin A1 (IgA1). Here, we describe a series of nano-liquid chromatography (LC)-mass spectrometry (MS) analyses that demonstrate the range of glycosyltransferase enzymatic activities involved in the biosynthesis of clustered O-glycans on IgA1. By utilizing nano-LC-MS relative quantitation of in vitro reaction products, our results provide unique insights into the biosynthesis of clustered IgA1 O-glycans. We have developed a workflow to determine glycoform-specific apparent rates of a human UDP-N-acetylgalactosamine:polypeptide N-acetylgalactosaminyltrasnfersase (GalNAc-T EC 2.4.1.41) and demonstrated how pre-existing glycans affect subsequent activity of glycosyltransferases, such as core 1 galactosyltransferase and α2,3- and α2,6-specific sialyltransferases, in successive additions in the biosynthesis of clustered O-glycans. In the context of IgA1, these results have potential to provide insight into the molecular mechanisms implicated in the pathogenesis of IgA nephropathy, an autoimmune renal disease involving aberrant IgA1 O-glycosylation. In a broader sense, these methods and workflows are applicable to the studies of the concerted and competing functions of other glycosyltransferases that initiate and extend mucin-type core 1 clustered O-glycosylation.
regulovaných látek z PET v potravinových simulantech kapalinovou -- chromatografií s hmotnostní detekcí (LC-MS /MS) 17 -- 3.1.11 Mikrobiologický stav a rizika papírových obalů z recyklovaných surovin 18 -- 3.1.12 21 -- 3.1.16 Stanovení SPF metodami in vivo a in vitro - srovnání výsledků .22 -- 3.1.17 Expozice nano-částicím
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Výroční zpráva se zaměřuje na čerpání podpory na rozvoj výzkumu ve Státním zdravotním ústavu v roce 2014. Určeno odborné veřejnosti.
- MeSH
- akademie a ústavy dějiny ekonomika MeSH
- biomedicínský výzkum dějiny ekonomika MeSH
- dějiny 21. století MeSH
- finanční podpora výzkumu jako téma dějiny MeSH
- vládní organizace MeSH
- Check Tag
- dějiny 21. století MeSH
- Publikační typ
- výroční zprávy MeSH
- Geografické názvy
- Česká republika MeSH
Mitochondria are exposed to reactive nitrogen species under physiological conditions and even more under several pathologic states. In order to reveal the mechanism of these processes we studied the effects of peroxynitrite on isolated beef heart mitochondria in vitro. Peroxynitrite has the potential to nitrate protein tyrosine moieties, break the peptide bond, and eventually release the membrane proteins into the solution. All these effects were found in our experiments. Mitochondrial proteins were resolved by 2D electrophoresis and the protein nitration was detected by immunochemical methods and by nano LC-MS/MS. Mass spectrometry confirmed nitration of ATP synthase subunit beta, pyruvate dehydrogenase E1 component subunit beta, citrate synthase and acetyl-CoA acetyltransferase. Immunoblot detection using chemiluminiscence showed possible nitration of other proteins such as cytochrome b-c1 complex subunit 1, NADH dehydrogenase [ubiquinone] iron-sulfur protein 2, elongation factor Tu, NADH dehydrogenase [ubiquinone] flavoprotein 2, heat shock protein beta-1 and NADH dehydrogenase [ubiquinone] iron-sulfur protein 8. ATP synthase beta subunit was nitrated both in membrane and in fraction prepared by osmotic lysis. The high sensitivity of proteins to nitration by peroxynitrite is of potential biological importance, as these enzymes are involved in various pathways associated with energy production in the heart.
- MeSH
- dusík metabolismus MeSH
- kyselina peroxydusitá farmakologie MeSH
- mitochondriální proteiny účinky léků metabolismus MeSH
- proteomika * MeSH
- skot MeSH
- srdeční mitochondrie účinky léků enzymologie metabolismus MeSH
- techniky in vitro MeSH
- tyrosin analogy a deriváty metabolismus MeSH
- zvířata MeSH
- Check Tag
- skot MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
