In this work, we present the synthesis and application of fluorescent rhodamine B hydrazide for the derivatization of simple oligosaccharides and complex glycans using a hydrazone formation chemistry approach. The labeling conditions and the experimental setup of CE/LIF were optimized by analyzing oligosaccharide standards. The CE/LIF separations were performed in polybrene-coated capillaries eliminating the need for the purification step after derivatization. The addition of methanol to the background electrolyte significantly increased the LIF detection sensitivity reaching the limits of detection in the attomole range. The resolution of carbohydrate samples was improved by using long (98 cm) capillaries and polymer additives (polybrene). The developed method was applied for CE/LIF and CE-MS analysis of N-linked glycans released from bovine ribonuclease B and the therapeutic monoclonal antibody of trastuzumab.
- Klíčová slova
- Capillary electrophoresis, Fluorescence, Glycan, Labeling, Mass spectrometry, Rhodamine B hydrazide,
- MeSH
- elektroforéza kapilární * metody MeSH
- fluorescenční barviva * chemie MeSH
- fluorescenční spektrometrie metody MeSH
- hmotnostní spektrometrie metody MeSH
- lasery MeSH
- oligosacharidy * chemie analýza MeSH
- polysacharidy * analýza chemie MeSH
- rhodaminy * chemie MeSH
- skot MeSH
- trastuzumab chemie analýza MeSH
- zvířata MeSH
- Check Tag
- skot MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- fluorescenční barviva * MeSH
- oligosacharidy * MeSH
- polysacharidy * MeSH
- rhodamine B MeSH Prohlížeč
- rhodaminy * MeSH
- trastuzumab MeSH
Timely identification of highly pathogenic bacteria is crucial for efficient mitigation of the connected harmful health effects. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) of intact cells enables fast identification of the microorganisms based on their mass spectrometry protein fingerprint profiles. However, the MALDI-TOF MS examination must be preceded by a time-demanding cultivation of the native bacteria to isolate representative cell samples to obtain indicative fingerprints. Isoelectric focusing (IEF) is capable of separating bacterial cells according to their isoelectric point while effectively removing other non-focusing compounds from sample matrix. In this work, we present a divergent-flow IEF chip (DF-IEF chip) fractionation as an alternative way for sample clean-up and concentration of bacterial cells to prepare samples usable for following MALDI-TOF MS analysis without the need of time-demanding cultivation. By means of DF-IEF chip method, we processed four species of highly pathogenic bacteria (Bacillus anthracis, Brucella abortus, Burkholderia mallei, and Yersinia pestis) inactivated with H2O2 vapors or by heat treatment at 62.5°C for 24 h. The DF-IEF chip method continually separated and concentrated the inactivated bacterial cells for subsequent detection using MALDI-TOF MS. The content of the inactivated bacteria in the DF-IEF chip fractions was evaluated with the MS analysis, where inactivated Y. pestis was found to be the most efficiently focusing species. Sensitivity analysis showed limits as low as 2 × 105 colony forming units per mL for inactivated B. anthracis.
- Klíčová slova
- biological agents, chip, fractionation, isoelectric focusing, pathogenic bacteria, whole cell separation,
- MeSH
- Bacteria izolace a purifikace chemie MeSH
- isoelektrická fokusace * metody MeSH
- peroxid vodíku chemie MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice * metody MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- peroxid vodíku MeSH
INTRODUCTION: Cyanobacterium Arthrospira platensis (AP) (Nordstedt) Gomont contains high content of phycobiliproteins (PBP), which are an important source for food industry. Methods effectively extracting proteins contained in AP cells are demanded to provide a supply of the material. Water-based extraction methods are advisable due to the high solubility of the PBP. OBJECTIVES: Extraction techniques such as ultrasound assisted extraction (UAE) and pressurized water extraction (PWE) are popular due to their environmental friendliness, better extraction efficiency, and faster extraction process. In this paper, efficiency of the two methods is compared. MATERIALS AND METHODS: PWE along with UAE is utilized for release of PBP from the AP cells. The extraction parameters including time, temperature, pressure, and ultrasound intensity are tested to obtain the most efficient setup. The methods were evaluated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and the replicates of PWE extracts were further analyzed by capillary isoelectric focusing with laser-induced fluorescence (cIEF-LIF). RESULTS: The developed PWE method using higher pressure treatment at lower temperature was significantly faster than UAE methods, and the SDS-PAGE results showed a high content of phycobiliproteins in the extracts. cIEF-LIF analysis showed that the sequential PWE of individual samples was repeatable, and the mild extraction provided a fluorescent profile similar to the commercially available C-phycocyanin standard. CONCLUSION: Pressurized water extraction was shown to be an efficient, rapid, and well-automated extraction method for AP proteins in general, including bioactive phycobiliproteins. Obtained results encourage the use of PWE in small-scale analytical applications for primary extraction of proteins.
- Klíčová slova
- Arthrospira platensis, phycocyanin, pressurized water extraction, spirulina, ultrasound assisted extraction,
- Publikační typ
- časopisecké články MeSH
BACKGROUND: Successful implementation of capillary isoelectric focusing (cIEF) in general requires the use of reference points - markers of isoelectric point. The low-molecular-mass fluorescent compounds based on the fluorescein structure, capable of marking distinct isoelectric points (pIs) with good focusing ability, stability, high extinction coefficient, and fluorescence are an alternative to the peptide and protein pI markers. RESULTS: Four synthetic pathways were used to produce the ampholytic compounds and the structures along with their basic physical and chemical properties are given. Previously published low-molecular-mass pI markers were used to calibrate the candidate fluorescent compounds using capillary IEF (cIEF) with laser-induced fluorescence (LIF) detection. Linear and point-to-point pI calibration approach were compared for the pI evaluation. The results show 21 fluorescent pI markers (FPIMs). This group of markers covers pH range from 3.10 to 10.21. The usability of the developed FPIMs was further demonstrated by analysis of fluorescein isothiocyanate (FITC) labeled immunoglobin (IgG) standard. SIGNIFICANCE AND NOVELTY: pI markers are crucial for tracking the pH gradient and pI determination of biological ampholytic compounds. This paper presents and characterizes a novel pI marker set capable of tracing isoelectric points and supporting highly sensitive LIF detection-based applications. A basis of a newly developed cIEF-based fractionation method is described as well.
- Klíčová slova
- Capillary isoelectric focusing, Isoelectric point marker, Laser-induced fluorescence, Protein characterization,
- MeSH
- elektroforéza kapilární metody MeSH
- fluorescein * chemie MeSH
- fluorescence MeSH
- fluorescenční barviva chemie MeSH
- fluorescenční spektrometrie MeSH
- isoelektrická fokusace * metody MeSH
- izoelektrický bod MeSH
- kapilární izoelektrická fokusace MeSH
- koncentrace vodíkových iontů MeSH
- lasery * MeSH
- molekulová hmotnost MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- fluorescein * MeSH
- fluorescenční barviva MeSH
In this study, the molecular interactions of the allylamine-type fungicide butenafine and a set of substructures ("fragments") with liposomes mimicking biological membranes were studied to gain a better understanding of the structural factors governing membrane affinity and perturbation. Specifically, drug/fragment-membrane interactions were investigated using an interdisciplinary approach involving micro differential scanning calorimetry, open-tubular capillary electrochromatography, nanoplasmonic sensing, and quartz crystal microbalance. By incubating the drug and the fragment compounds with liposomes with varying lipid composition or by externally adding the compounds to preformed liposomes, a detailed mechanistic picture on the underlying drug/fragment-membrane interactions was obtained. The nature and the degree of ionisation of polar head groups of the lipids had a major influence on the nature of drug-membrane interactions, and so had the presence and relative concentration of cholesterol within the membranes. The in-depth understanding of drug/fragment-membranes interactions established by the presented interdisciplinary fragment-based approach may be useful in guiding the design and early-stage evaluation of prospective antifungal drug candidates, and the discovery of agents with improved membrane penetrating characteristics in general.
- MeSH
- biomimetické materiály chemie farmakologie MeSH
- cholesterol chemie MeSH
- fungicidy průmyslové * chemie farmakologie MeSH
- liposomy * chemie MeSH
- mikrorovnovážné techniky křemenného krystalu MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- cholesterol MeSH
- fungicidy průmyslové * MeSH
- liposomy * MeSH
We present a set of novel low-molecular-mass (LMM) compounds possessing ampholytic properties. The compounds were designed to perform as markers of isoelectric point (pI) in different isoelectric focusing (IEF) formats and feature direct detectability in UV and visible wavelength regions. Capillary isoelectric focusing (cIEF) was used to determine the purity of the focusing species and the compounds' pI values. Nitrophenol-based pI markers (NPIMs) published previously were used as standards for the pI value calibration. The presented compounds focused very well, but small portion of them contained focusing impurities, thus, we recommend them for use in other IEF formats like gel IEF and preparative IEF. Moreover, multi-wavelength detection enabled determination of individual markers based on their specific spectral profiles and different absorption at selected detection wavelengths in the electropherogram. The presented compounds compose a group of chemicals featuring excellent shelf stability and isoelectric focusing properties, inexpensive synthesis, universal/multimode detectability, and good solubility at pI. The presented results provide a solid ground for their use as reference standards in various isoelectric focusing methods.
- Klíčová slova
- Capillary, Colored, Focusing, Isoelectric point, Marker,
- MeSH
- isoelektrická fokusace metody MeSH
- izoelektrický bod MeSH
- koncentrace vodíkových iontů MeSH
- protonmotorická síla * MeSH
- pufry MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- pufry MeSH
We have developed a planar chip utilizing divergent geometry of separation channel capable of vertical free-flow electrophoresis of particles at flows of lower hundreds of microliters per minute. The divergent flow isoelectric focusing (DF-IEF) chip consists of two sheets of clear polystyrene glass which serve as a base with working channels and a top cover sealing the separation channel. Optimization showed that the chip is capable to form pH gradient within 1 h and separation is completed in 5 or more minutes depending on the sample volume. The vertical position of the chip enabled analysis of sedimenting particles including microorganisms. Four different common bacteria species inactivated with H2O2 vapors were analyzed in a series of experiments. Isoelectric points were determined with capillary isoelectric focusing with following fractionation using DF-IEF with intact cell matrix-assisted laser desorption/ionization mass spectrometry detection. The DF-IEF chip fractionation proved promising for bacterial sample preparation from complex matrices for subsequent identification of whole cells by mass spectrometry.
- Klíčová slova
- Bacteria, Chip, Intact cell, Isoelectric focusing, Mass spectrometry,
- MeSH
- Bacteria MeSH
- chemická frakcionace * MeSH
- isoelektrická fokusace MeSH
- peroxid vodíku * MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- peroxid vodíku * MeSH
In this work, we characterize a previously synthesized multi-cationic aminopyrene-based labeling tag for oligosaccharide analysis by capillary electrophoresis with laser-induced fluorescence detection (CE/LIF). The fluorescent tag, 4,4',4''-(8-aminopyrene-1,3,6-trisulfonyl)tris(1-methylpiperazine) (APTMP), was characterized by reaction with standard maltooligosaccharides and the labeling parameters such as fluorescent tag concentration, labeling temperature, and time as well as influence of a reducing agent and its solvent were investigated in terms of labeling efficiency. The nanomolar limit of detection of CE/LIF analysis of APTMP labeled maltopentaose was determined. However, significant amount of the oligosaccharides was reduced to alditols, which negatively affects the yield and rate of the labeling reaction. Under optimized conditions, a highly reproducible labeling by multi-cationic APTMP was obtained; however, the most commonly used labeling by multi-anionic 8-aminopyrene-1,3,6-trisulfonic acid trisodium salt (APTS) is superior compared to APTMP labeling. Lower reactivity of APTMP compared to APTS can be explained by the loss of nucleophilicity induced by substitution of the sulfonate groups with more electron-withdrawing aminosulfonyl ones. On contrary, APTMP is still a promising tag for oligosaccharide labeling followed by CE-MS in a positive ion mode, which is considered to be more sensitive than MS detection of APTS in a negative ion mode.
- Klíčová slova
- Capillary electrophoresis, Fluorescence, Labeling, Oligosaccharides, Reductive amination,
- MeSH
- elektroforéza kapilární * MeSH
- kationty MeSH
- lasery MeSH
- oligosacharidy MeSH
- pyreny MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- kationty MeSH
- oligosacharidy MeSH
- pyreny MeSH
We present a novel method for concentration and purification of DNA from biological samples. The method is based on isotachophoretic separation of DNA strands in a separation bed made of a disposable nonwoven fabric strip. Application of oxalate as the leading ion prevented corrosion of the carbon anode and also the leading ion was continually removed from the system due to its decomposition into CO2 at the anode. The fractions were marked by three colored markers of electrophoretic mobility closely surrounding the mobility of DNA. The fraction collection was realized by a centrifugal drain of cut out strip segments. The method was evaluated using two purified salmon sperm DNA fragments of lengths 200 bp and 2000 bp. The results confirmed the high DNA concentrating effect of the method (34-fold increase of the original DNA concentration). The composition of running solutions and voltage program were optimized in order to finish the analysis within 30 min. The optimized method was used to extract, concentrate and purify DNA from a crude yeast cell lysate. The maximum DNA enrichment factor decreased to 12 due to the stretching of DNA zones caused by low-molecular contaminants present in the original lysate. The average recovery determined for yeast DNA was 71 ± 11% (n = 3). The connected elimination of the proteins from DNA zones resulted in the purification factor value of 582 for DNA vs proteins. This demonstrates that the presented method is capable to concentrate DNA from the bulk volume and to further purify it from crude cell lysates using a simple instrumentation and low-cost disposable separation bed.
- Klíčová slova
- DNA, Electrophoretic mobility, Isotachophoresis, Purification,
- MeSH
- DNA fungální izolace a purifikace MeSH
- izotachoforéza přístrojové vybavení metody MeSH
- Saccharomyces cerevisiae chemie cytologie MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- DNA fungální MeSH
In this work, we compare labeling by two negatively charged fluorescent labels, 8-aminopyrene-1,3,6-trisulfonic acid (APTS) and 8-(2-hydrazino-2-oxoethoxy)pyrene-1,3,6-trisulfonic acid (Cascade Blue hydrazide [CBH]). Effectiveness of the labeling chemistries were investigated by 4-hydroxybenzaldehyde and maltoheptaose followed by LC/UV-MS and CE/LIF analysis, respectively. The reaction yield of APTS labeling was determined to be only ∼10%. This is due to reduction of almost 90% of the analyte by sodium cyanoborohydride to alcohol, which cannot be further labeled via reductive amination. However, the CBH labeling provides ∼90% reaction yield based on the LC/UV-MS measurements. The significantly higher labeling yield was also confirmed by CE/LIF measurements. Finally, the more effective hydrazone formation technique of CBH was characterized and applied for N-linked glycan analysis by CE/LIF.
- Klíčová slova
- Hydrazone formation, Labeling, Oligosaccharides, Reductive amination,
- MeSH
- aminace MeSH
- chromatografie kapalinová metody MeSH
- elektroforéza kapilární metody MeSH
- fluorescenční barviva chemie MeSH
- hmotnostní spektrometrie metody MeSH
- hydrazony chemie MeSH
- oligosacharidy analýza chemie MeSH
- pyreny chemie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- 1-aminopyrene-3,6,8-trisulfonic acid MeSH Prohlížeč
- fluorescenční barviva MeSH
- hydrazony MeSH
- oligosacharidy MeSH
- pyreny MeSH
