Evaluation of solid-phase extraction sorbents for purification of oligosaccharides and glycans derivatized by positively charged labels followed by capillary electrophoretic analysis.

The sample preparation including labeling and clean-up represents a key analytical step in the analysis of oligosaccharides and glycans by either chromatographic or electrophoretic separation methods. Although the majority of labeling has been performed by neutral and/or negatively charged tags, the introduction of a positive charge into the saccharide molecule can significantly improve the analysis, especially with mass spectrometry detection. In this work, we present the evaluation of five solid-phase extraction sorbents differing in extraction chemistry for the clean-up and concentration of positively labeled maltooligosaccharides from the reaction mixtures. Maltooligosaccharides containing four to seven glucose units were labeled by cationic tags (2-aminoethyl)trimethylammonium chloride and (carboxymethyl)trimethylammonium chloride hydrazide and the extraction conditions were optimized followed by electrophoretic analysis with conductivity detection. The effects of the solid-phase extraction sorbent chemistry, extraction conditions, and sample composition are discussed. All tested sorbents were capable of cleaning up maltooligosaccharides from the reaction mixtures to some extent after optimization of the solid-phase extraction procedure (51.9%-98.9% recovery). The best-rated amide-based sorbent was used to process the sample of N-linked glycans enzymatically released from ribonuclease B.

Fabrication of monolithic capillary columns with inner diameter 50-530 µm employing a mixture of pentaerythritol tetraacrylate and polyhedral oligomeric silsesquioxane-methacrylate as crosslinkers.

Highly crosslinked monolithic capillary columns with inner diameters in the range of 50-530 µm were prepared by radical polymerization of pentaerythritol tetraacrylate, polyhedral oligomeric silsesquioxane-methacrylate, and n-octadecyl methacrylate in the presence of methanol, dodecyl alcohol, and polyethylene glycol lauryl ether. Columns were evaluated by inverse size-exclusion chromatography employing a set of polystyrene standards of narrow molecular-size distribution and by scanning electron microscopy. Chromatographic performance under reversed-phase conditions was also evaluated. The combination of two effective crosslinkers as pentaerythritol tetraacrylate and polyhedral oligomeric silsesquioxane-methacrylate in the polymerization mixture allows for the preparation of robust and efficient monolithic capillary columns within a fairly wide range of internal diameters.

No-additive salting-out liquid-liquid extraction-A tool for purification of positively charged compounds from highly salted reaction mixtures.

A novel and original application of salting-out assisted liquid-liquid extraction is presented. This technique was used to purify the final reaction products (quaternary ammonium salts) from unreacted components and by-products present in multiple excesses. The partition of two structurally related compounds as (2-aminoethyl)trimethylammonium salt (a labeling reagent) and a derivative of [2-(imidazoline-1-yl)ethyl]trimethylammonium salt (a final reaction product of N-acetylglucosamine labeling by (2-aminoethyl)trimethylammonium salt) between acetonitrile-rich and water-rich layers was monitored by hydrophilic interaction chromatography with electrospray ionization mass spectrometry. Despite the poor solubility of both highly polar substances in solutions containing a high concentration of acetonitrile, the main portion of the labeling reagent (72%) can be removed from the crude reaction mixture in the first extraction step using 95% acetonitrile/5% water as an extraction solvent. The purified final reaction product contained only 2% of the labeling reagent, and it was suitable for analysis by direct infusion mass spectrometry to confirm its identity. The capability of the suggested purification protocol to process small-volume highly salted reaction mixtures was also proven by analysis of saccharide mixture containing glucose, maltose, and maltotriose labeled by the positively charged tag.

Monoliths in capillary electrochromatography and capillary liquid chromatography in conjunction with mass spectrometry.

Here, we have reviewed separation studies utilizing monolithic capillary columns for separation of compounds preceding MS analysis. The review is divided in two parts according to the used separation method, namely CEC and capillary LC (cLC). Based on our overview, monolithic CEC-MS technique have been more focused on the syntheses of highly specialized and selective separation phase materials for fast and efficient separation of specific types of analytes. In contrast, monolithic cLC-MS is more widely used and is often employed, for instance, in the analysis of oligonucleotides, metabolites, and peptides and proteins in proteomic studies. While poly(styrene-divinylbenzene)-based and silica-based monolithic capillaries found their place in proteomic analyses, the other laboratory-synthesized monoliths still wait for their wider utilization in routine analyses. The development of new monolithic materials will most likely continue due to the demand of more efficient and rapid separation of increasingly complex samples.

SDS-PAGE and Gel IEF: Tool for Differentiation of Methicillin-Resistant and Methicillin-Sensitive Strains of Staphylococcus aureus.

The methicillin-resistant Staphylococcus aureus causes difficult-to-treat healthcare-associated infections in humans. For fast and effective selection of an appropriate antibiotic therapy, it is essential to have rapid and reliable methods for differentiation of methicillin-resistant S. aureus from less dangerous methicillin-sensitive S. aureus. There have been many methods for the identification of methicillin-resistant S. aureus described but none has been accepted as an international standard. The most commonly used techniques such as phenotyping and genotyping have a few disadvantages, for instance, these techniques are not reproducible and stable. In addition, they are time-consuming, expensive, and they are not capable to distinguish all S. aureus strains. In this study, the methicillin-resistant and methicillin-sensitive S. aureus isolates obtained from patients were extracted in hot water. The released proteins were characterised by sodium dodecyl sulphate polyacrylamide gel electrophoresis and gel isoelectric focusing. These two methods were able to differentiate among tested bacterial strains. The proposed methods are time saving, they are applicable in standard biochemical laboratories, and they do not require any expensive equipment.

Use of electrophoretic techniques and MALDI-TOF MS for rapid and reliable characterization of bacteria: analysis of intact cells, cell lysates, and "washed pellets".

In this study electrophoretic and mass spectrometric analysis of three types of bacterial sample (intact cells, cell lysates, and "washed pellets") were used to develop an effective procedure for the characterization of bacteria. The samples were prepared from specific bacterial strains. Five strains representing different species of the family Rhizobiaceae were selected as model microorganisms: Rhizobium leguminosarum bv. trifolii, R. leguminosarum bv. viciae, R. galegae, R. loti, and Sinorhizobium meliloti. Samples of bacteria were subjected to analysis by four techniques: capillary zone electrophoresis (CZE), capillary isoelectric focusing (CIEF), gel IEF, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). These methods are potential alternatives to DNA-based methods for rapid and reliable characterization of bacteria. Capillary electrophoretic (CZE and CIEF) analysis of intact cells was suitable for characterization of different bacterial species. CIEF fingerprints of "washed pellets" and gel IEF of cell lysates helped to distinguish between closely related bacterial species that were not sufficiently differentiated by capillary electrophoretic analysis of intact cells. MALDI-TOF MS of "washed pellets" enabled more reliable characterization of bacteria than analysis of intact cells or cell lysates. Electrophoretic techniques and MALDI-TOF MS can both be successfully used to complement standard methods for rapid characterization of bacteria.

Sulfate supplementation affects nutrient and photosynthetic status of Arabidopsis thaliana and Nicotiana tabacum differently under prolonged exposure to cadmium.

Hydroponic experiments were performed to examine the effect of prolonged sulfate limitation combined with cadmium (Cd) exposure in Arabidopsis thaliana and a potential Cd hyperaccumulator, Nicotiana tabacum. Low sulfate treatments (20 and 40 µM MgSO4) and Cd stress (4 µM CdCl2) showed adverse effects on morphology, photosynthetic and biochemical parameters and the nutritional status of both species. For example, Cd stress decreased NO3- root content under 20 µM MgSO4 to approximately 50% compared with respective controls. Interestingly, changes in many measured parameters, such as chlorophyll and carotenoid contents, the concentrations of anions, nutrients and Cd, induced by low sulfate supply, Cd exposure or a combination of both factors, were species-specific. Our data showed opposing effects of Cd exposure on Ca, Fe, Mn, Cu and Zn levels in roots of the studied plants. In A. thaliana, levels of glutathione, phytochelatins and glucosinolates demonstrated their distinct involvement in response to sub-optimal growth conditions and Cd stress. In shoot, the levels of phytochelatins and glucosinolates in the organic sulfur fraction were not dependent on sulfate supply under Cd stress. Altogether, our data showed both common and species-specific features of the complex plant response to prolonged sulfate deprivation and/or Cd exposure.

Divergent-flow isoelectric focusing for separation and preparative analysis of peptides.

A divergent-flow isoelectric focusing (DF IEF) technique has been applied for the separation and preparative analysis of peptides. The parameters of the developed DF IEF device such as dimension and shape of the separation bed, selection of nonwoven material of the channel, and separation conditions were optimized. The DF IEF device was tested by the separation of a peptide mixture originating from the tryptic digestion of BSA, cytochrome c, and myoglobin. The pH gradient of DF IEF was created by the autofocusing of tryptic peptides themselves without any addition of carrier ampholytes. The focusing process was monitored visually using colored pI markers, and the obtained fractions were analyzed by RP-HPLC and ESI/TOF-MS. DF IEF operating in the autofocusing mode provides an efficient preseparation of peptides, which is comparable with a commercially available MicroRotofor multicompartment electrolyzer and significantly improves sequence coverage of analyzed proteins. The potential of the DF IEF device as an efficient tool for the preparative scale separations was demonstrated by the isolation of caseinomacropeptide (CMP) from a crude whey solution.

Low-molecular-mass colored compounds for fine tracing of pH gradient on broad and narrow scale in isoelectric focusing.

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.

Characterization and applications of a trioctyl(3/4-vinylbenzyl)phosphonium stationary phase for use in capillary liquid chromatography.

The morphology, composition, and selectivity of a silica-based monolithic stationary phase, grafted by a layer of trioctyl(3/4-vinylbenzyl)phosphonium chloride ([P888VBn]Cl), is presented. The results of elemental analysis confirmed that the prepared stationary phase contains 38.8 at.% of silicon, 60.2 at.% of carbon, and 1.0 at.% of phosphorus. Capillary columns (150 × 0.1 mm) for liquid chromatography were evaluated using alkylbenzenes, monosubstituted benzenes, polyaromatic compounds, substituted benzene regioisomers, and aromatic carboxylic and amino acids. The prepared ionic liquid (IL)-based stationary phase exhibits hydrophobic, hydrophilic, and electrostatic interactions, as confirmed by experiments on the evaluation of the effect of the mobile phase composition (content of acetonitrile and ammonium formate) on the isocratic chromatographic separation. Thus, the IL-based capillary column demonstrates a unique separation selectivity compared to Phenyl-, C8-, and C18-stationary phases, and high efficiency for an expanding number of structurally diverse compounds.

Separation of labeled isomeric oligosaccharides by hydrophilic interaction liquid chromatography - the role of organic solvent in manipulating separation selectivity of the amide stationary phase.

The advantages of using mixtures of organic solvents for the separation of labeled oligosaccharides on the amide stationary phase under hydrophilic interaction liquid chromatography conditions are presented. The effect of the type of buffer as well as solvent or their mixtures on retention of uracil, saccharide labeling reagents (2-aminobenzoic acid, 2-aminobenzamide, ethyl 4-aminobenzoate, procainamide), and corresponding labeled saccharides were evaluated. The successful isocratic separation of labeled isomeric trisaccharides (maltotriose, panose, and isomaltotriose) was achieved in the mobile phase consisting of a 90% (v/v) mixture of organic solvents (methanol/acetonitrile 60:40) and 10% (v/v) 30 mM ammonium formate, pH 3.3. Changing the volume ratio between methanol/acetonitrile from 60:40 to 50:50 (v/v) allowed to obtain the separation of di-, tri-, and tetrasaccharides labeled by ethyl 4-aminobenzoate in less than 10.5 min.

Pressurized water extraction - the fast and efficient method for isolation of bioactive proteins from Viscum album leaves.

New strategies for the fast, efficient, and environmentally friendly extraction of proteins are required to isolate desired bioactive compounds from a technological point of view. In this study, utilization of the pressurized water extraction (PWE) at low temperature (40 °C) for isolation of mistletoe proteins was investigated. PWE effectiveness, based on protein fingerprints, were compared with those obtained by conventional extractions using 10 mmol L-1 Tris-HCl buffer pH 8.3, 50 mmol L-1 phosphate buffer pH 7, or deionized water. The extracts were precipitated using acetone, trichloroacetic acid (TCA), and 20% (w/v) TCA/acetone and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. PWE was more or equally efficient for isolation of mistletoe proteins than evaluated conventional extraction methods. The proteomic analysis combining mass spectrometry and database searching confirmed the presence of 35 proteins in PWE extracts precipitated by acetone, which was the most compounds identified from all studied extracts. The PWE high extraction power was revealed for multiple viscotoxin isoforms and specific enzymes indispensable for the synthesis of terpenes.

DNA purification and concentration by isotachophoresis in nonwoven fabric strip.

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.

Capillary electromigration separation of proteins and microorganisms dynamically modified by chromophoric nonionogenic surfactant.

A chromophoric nonionogenic surfactant poly(ethylene glycol) 3-(2-hydroxy-5-n-octylphenylazo)-benzoate, HOPAB, has been prepared and used as a buffer additive for a dynamic modification of proteins and/or microorganisms including Escherichia coli , Staphylococcus epidermidis (biofilm-positive and biofilm-negative), and the strains of yeast cells Candida albicans and Candida parapsilosis (biofilm-positive and biofilm-negative) during a capillary electrophoresis and a capillary isoelectric focusing (CIEF) with UV detection at 326 nm. Values of isoelectric points of labeled proteins and microorganisms have been calculated using UV-detectable pI markers and have been found comparable with pI of the native compounds. Minimum detectable amount has been assessed lower than picograms of proteins and lower than a hundred cells injected into a separation capillary. The introduced labeling method facilitates CIEF separation of microorganisms from the clinical sample of the infected urine at their clinically important levels in the pH gradient pH range of 2-5 and their subsequent cultivation. At the same time, it has enabled the determination of albumin in human urine as a major clinical marker of urinary tract infections and kidney diseases.

Low-molecular-mass nitrophenol-based compounds suitable for the effective tracking of pH gradient in isoelectric focusing.

Low-molecular-mass isoelectric point (pI) markers are a reasonable alternative to commonly used peptide and protein pI markers in the field of isoelectric focusing. We present a comparative study dealing with characterization of 44 nitrophenol-based compounds synthesized by the Mannich reaction which pI values cover a pH range from 3.2 to 10.4. The values of pIs of all the presented nitrophenol-based compounds were precisely determined by capillary isoelectric focusing technique (cIEF) when a standard deviation of measurement reached a value of a few hundredths of a pH unit (n ≥ 3). Differences have been found between in silico analysis of acid-base properties of the investigated compounds and the experimental cIEF data especially at the basic pH region. The data from the acidic pH range showed better correlation between these methods. Only three compounds were excluded from the group of pI markers because they contained impurities originating from the synthesis or long-term storage. Based on the presented results we identified 41 nitrophenol-based pI markers (NPIMs) which can fulfill requirements of the most applications in a field of isoelectric focusing analyses.

Immobilization of a phosphonium ionic liquid on a silica monolith for hydrophilic interaction chromatography.

A methodology for preparing phosphonium-based ionic liquid modified silica-based monolithic capillary columns is presented. The silica monolithic columns with dimensions of 150 × 0.1 mm were modified by a phosphonium-based ionic liquid (trioctyl(3/4-vinylbenzyl)phosphonium chloride) via 3-(trimethoxysilyl)propyl methacrylate. The prepared columns were evaluated under hydrophilic interaction liquid chromatography separation conditions, employing a sample mixture containing purine and pyrimidine bases and nucleosides. Detection was made by UV. The high efficiency of the original silica monolith was preserved even after modification, and it reached values in the range of 98,000-174,000 theoretical plates/m. The effects of the concentration of acetonitrile in the mobile phase, the presence of additives in the mobile phase, such as, acetic acid or ammonium acetate, and the pH of the mobile phase on the separation of some selected analytes were investigated. The prepared columns showed different separation selectivity compared to silica, phenyl and sulfobetaine stationary phases.

Bridged polysilsesquioxane-based wide-bore monolithic capillary columns for hydrophilic interaction chromatography.

The synthesis and characterization of large-bore silica-based monolithic capillary columns (0.32mm×150mm) are presented. Columns were prepared by acidic hydrolysis of a mixture containing tetramethoxysilane (TMOS) and 1,2-bis(trimethoxysilyl)ethane (BTME) in different molar ratios in the presence of polyethylene glycol and urea. The monoliths were modified by zwitterionic monomer [2-(methacryloyloxy)ethyl]-dimethyl-(3-sulfopropyl)-ammonium hydroxide via 3-(trimethoxysilyl)propyl methacrylate. Prepared stationary phases were evaluated by scanning electron microscopy and chromatographic separation of nucleobases and their derivatives in the HILIC mode. The best chromatographic results were obtained with the column prepared from the reaction mixture containing BTME and TMOS in a 1:4 molar ratio. The permeability of such column reached 1.68×10-14m2 and the efficiency, expressed as a height equivalent of the theoretical plate, did not exceed 10.5µm for the tested compounds. The columns were successfully applied to HILIC separation of native and labeled oligosaccharides and glycans released from bovine ribonuclease B and human immunoglobulin G.

Polymetacrylate and hybrid interparticle monolithic columns for fast separations of proteins by capillary liquid chromatography.

Preparation of organic polymer monolithic columns in fused silica capillaries was aimed at fast gradient separation of proteins. For this purpose, polymerization in situ procedure was optimized, using ethylene dimetacrylate and butyl metacrylate monomers with azobisisobutyronitrile as initiator of the polymerization reaction in presence of non-aqueous porogen solvent mixtures composed of 1-propanol and 1,4-butanediol. The separation of proteins in totally monolithic capillary columns was compared with the chromatography on a new type of "hybrid interparticle monolithic" capillary columns, prepared by in situ polymerization in capillary packed with superficially porous spherical beds, 37-50 microm. The "hybrid" columns showed excellent stability and improved hydrodynamic flow properties with respect to the "totally" monolithic capillary columns. The separation selectivity is similar in the two types of columns. The nature of the superficially porous layer (bare silica or bonded C18 ligands) affects the separation selectivity less significantly than the porosity (density) of the monolithic moiety in the interparticle space, controlled by the composition of the polymerization mixture. The retention behaviour of proteins on all prepared columns is consistent with the reversed-phase gradient elution theory.

Instrument platforms for nano liquid chromatography.

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.

Phosphatidylcholine covalently linked to a methacrylate-based monolith as a biomimetic stationary phase for capillary liquid chromatography.

In this study a strategy to immobilize phospholipids onto a polymer-based stationary phase is described. Methacrylate-based monoliths in capillary format (150×0.1mm) were modified by soybean phosphatidylcholine through 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide coupling to obtain stationary phases suitable to mimic cell surface membranes. The covalent coupling reaction involves the phosphate group in phospholipids; therefore, the described methodology is suitable for all types of phospholipids. Immobilization of soy bean phosphatidylcholine on the monolith was confirmed by attenuated total reflectance Fourier transform infrared spectroscopy and gas chromatography-mass spectrometry of the fatty alcohol profile, generated upon reductive cleavage of the fatty acyl side chains of the phospholipid on the monolith surface with lithium aluminium hydride. The prepared stationary phases were evaluated through studies on the retention of low-molar mass model analytes including neutral, acidic, and basic compounds. Liquid chromatographic studies confirmed predominant hydrophobic interactions between the analytes and the synthesized stationary phase; however, electrostatic interactions contributed to the retention as well. The synthesized columns showed high stability even with fully aqueous mobile phases such as Dulbecco's phosphate-buffered saline solution.