Protein glycosylation analysis is challenging due to the structural variety of complex conjugates. However, chromatographically separating glycans attached to tryptic peptides enables their ...site-specific characterization. For this purpose, we have shown the importance of selecting a suitable hydrophilic interaction liquid chromatography (HILIC) stationary phase in the separation of glycopeptides and their isomers. Three different HILIC stationary phases, i.e., HALO
penta-HILIC, Glycan ethylene bridged hybrid (BEH) Amide, and ZIC-HILIC, were compared in the separation of complex
-glycopeptides of hemopexin and Immunoglobulin G glycoproteins. The retention time increased with the polarity of the glycans attached to the same peptide backbone in all HILIC columns tested in this study, except for the ZIC-HILIC column when adding sialic acid to the glycan moiety, which caused electrostatic repulsion with the negatively charged sulfobetaine functional group, thereby decreasing retention. The HALO
penta-HILIC column provided the best separation results, and the ZIC-HILIC column the worst. Moreover, we showed the potential of these HILIC columns for the isomeric separation of fucosylated and sialylated glycoforms. Therefore, HILIC is a useful tool for the comprehensive characterization of glycoproteins and their isomers.
Saccharides form one of the major constituents of biological macromolecules in living organisms. Many biological processes including protein folding, stability, immune response and receptor ...activation are regulated by glycosylation. In this work, we optimized a capillary electrophoresis method with capacitively coupled contactless conductivity detection for the separation of eight monosaccharides commonly found in glycoproteins, namely D‐glucose, D‐galactose, D‐mannose, N‐acetyl‐D‐glucosamine, N‐acetyl‐D‐galactosamine, D‐fucose, N‐acetylneuraminic acid, and D‐xylose. A highly alkaline solution of 50 mM sodium hydroxide, 22.5 mM disodium phosphate, and 0.2 mM CTAB (pH 12.4) was used as a background electrolyte in a 10 µm id capillary. To achieve baseline separation of all analytes, a counter‐directional pressure of –270 kPa was applied during the separation. The limits of detection of our method were below 7 µg/ml (i.e., 1.5 pg or 1 mg/g protein) and the limits of quantification were below 22 µg/ml (i.e., 5 pg or 3 mg/g protein). As a proof of concept of our methodology, we performed an analysis of monosaccharides released from fetuin glycoprotein by acid hydrolysis. The results show that, when combined with an appropriate pre‐concentration technique, the developed method can be used as a monosaccharide profiling tool in glycoproteomics and complement the routinely used LC‐MS/MS analysis.
A typical bottom-up proteomic workflow comprises sample digestion with trypsin, separation of the hydrolysate using reversed-phase HPLC, and detection of peptides via electrospray ionization (ESI) ...tandem mass spectrometry. Despite the advantages and wide usage of protein identification and quantification, the procedure has limitations. Some domains or parts of the proteins may remain inadequately described due to inefficient detection of certain peptides. This study presents an alternative approach based on sample acetylation and mass spectrometry with atmospheric pressure chemical ionization (APCI) and atmospheric pressure photoionization (APPI). These ionizations allowed for improved detection of acetylated peptides obtained via chymotrypsin or glutamyl peptidase I (Glu-C) digestion. APCI and APPI spectra of acetylated peptides often provided sequence information already at the full scan level, while fragmentation spectra of protonated molecules and sodium adducts were easy to interpret. As demonstrated for bovine serum albumin, acetylation improved proteomic analysis. Compared to ESI, gas-phase ionizations APCI and APPI made it possible to detect more peptides and provide better sequence coverages in most cases. Importantly, APCI and APPI detected many peptides which passed unnoticed in the ESI source. Therefore, analytical methods based on chymotrypsin or Glu-C digestion, acetylation, and APPI or APCI provide data complementary to classical bottom-up proteomics.
•Characterization and comparison of three columns providing RP/AEX mechanism.•Effects of buffer pH and concentration on retention and peak shape were evaluated.•Contribution of ionic interactions ...strongly depends on the buffer pH and column type.•Fast, efficient peptides separation needs appropriate buffer pH and concentration.•Fourteen peptides were baseline separated on all the columns using buffer of pH 3.0.
In this work, two mixed-mode columns from a different manufacturers and one marketed as a reversed-phase column were characterized and compared in the terms of their interaction abilities, retentivity, peak symmetry, and applicability for peptide separation. All the tested columns contain octadecyl ligand and positively charged modifier, i.e. pyridyl group for the reversed-phase column XSelect CSH C18, quaternary alkylamine for mixed-mode column Atlantis PREMIER BEH C18 AX, and permanently charged moiety (details not available from the manufacturer) for mixed-mode column Luna Omega PS C18. For detailed characterization and comparison of their interaction potential, several approaches were used. First, a simple Walters test was performed to estimate hydrophobic and silanophilic interactions of the tested columns. The highest values of both parameters were observed for column Atlantis PREMIER BEH C18 AX. To investigate the effect of pH and buffer concentration on retention, mobile phases composed of acetonitrile and buffer (ammonium formate, pH 3.0; ammonium acetate pH 4.7 and pH 6.9) in various concentrations (5mM; 10mM; 15mM and 20mM) were used. The analysis of permanently charged compounds was used to describe the electrostatic interaction abilities of the stationary phases. The most significant contribution of electrostatic interactions to the retention was observed for Atlantis PREMIER BEH C18 AX column in the mobile phase with buffer of pH 3.0. A set of ten dipeptides, three pentapeptides and one octapeptide was used to investigate the effects of pH and buffer concentration on retention and peak symmetry. Each of the tested columns provides the optimal peak shape under different buffer pH and concentration. The gradient separation of the 14 tested peptides was used to verify the application potential of the tested columns for peptide separation. The best separation was achieved within 4 minutes on column Atlantis PREMIER BEH C18 AX.
Three chiral stationary phases were prepared by dynamic coating of sulfobutylether‐β‐cyclodextrin (SBE‐β‐CD) with different degrees of substitution, onto strong anion‐exchange stationary phases. The ...enantioselective potential and stability of newly prepared chiral stationary phases were examined using a set of structurally different chiral analytes. Measurements were performed in RP‐HPLC. Mobile phases consisted of methanol/formic acid, pH 2.10, and methanol/10 mM ammonium acetate buffer, pH 4.00, in various volume ratios. SBE‐β‐CDs with degrees of substitution (DS) 4, 6.3, and 10 proved suitable for the enantioseparation of 14, 11, and 8 analytes, respectively. The SBE‐β‐CD DS 4 based chiral stationary phase enabled the enantioseparation of the nearly all basic and neutral compounds. Chiral stationary phases with higher sulfobutylether‐β‐cyclodextrin substitution (especially DS 10) yielded higher enantioresolution values for acidic compounds.
Metallacarborane clusters, such as COSAN, belong to surface-active and low-coordinating nanosized anions. Therefore, they have been used as separate building blocks in self- and co-assembly. As a ...step forward, we synthesized via ring-opening metathesis polymerization a novel polyelectrolyte, poly(norbornene-COSAN), PNC, with metallacarborane anions covalently attached to a polynorbornene backbone. The resulting PNC, with the degree of polymerization around 120, is soluble in polar organic solvents, and it can be deposited on a substrate as separate polymer chains or as multichain aggregates, with the bundle-of-fibril patterning. PNC is miscible with polyethylene oxide (PEO), forming the PNC/PEO composite. As shown by solid-state nuclear magnetic resonance spectroscopy, Li+ counterions are firmly coordinated in the PNC matrix, exhibiting rather restricted dynamics. In contrast, the mixing of PNC with PEO leads to a substantial increase of Li+ dynamics. As the result, the mobility of Li+ in the PNC/PEO composite remains almost unrestrained, which makes the COSAN-containing polyelectrolytes promising candidates for ion-conducting materials.
Knowledge of the benefits of mTOR inhibition concerning adipogenesis and inflammation has recently encouraged the investigation of a new generation of mTOR inhibitors for non-alcoholic ...steatohepatitis (NASH). We investigated whether treatment with a specific mTORC1/C2 inhibitor (Ku-0063794; KU) exerted any beneficial impacts on experimentally-induced NASH in vitro and in vivo. The results indicated that KU decreases palmitic acid-induced lipotoxicity in cultivated primary hepatocytes, thus emerging as a successful candidate for testing in an in vivo NASH dietary model, which adopted the intraperitoneal KU dosing route rather than oral application due to its significantly greater bioavailability in mice. The pharmacodynamics experiments commenced with the feeding of male C57BL/6 mice with a high-fat atherogenic western-type diet (WD) for differing intervals over several weeks aimed at inducing various phases of NASH. In addition to the WD, the mice were treated with KU for 3 weeks or 4 months. Acute and chronic KU treatments were observed to be safe at the given concentrations with no toxicity indications in the mice. KU was found to alleviate NASH-related hepatotoxicity, mitochondrial and oxidative stress, and decrease the liver triglyceride content and TNF-α mRNA in at least one set of in vivo experiments. The KU modulated liver expression of selected metabolic and oxidative stress-related genes depended upon the length and severity of the disease. Although KU failed to completely reverse the histological progression of NASH in the mice, we demonstrated the complexity of mTORC1/C2 signaling regulation and suggest a stratified therapeutic management approach throughout the disease course.
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Statin therapy should be considered in children with familial hypercholesterolemia and sustained high LDL-C levels. There are no data on rosuvastatin exposure in patients <6 years and efficacy/safety ...can only be derived from case reports. Our aim was to examine developmental changes in pharmacokinetics of rosuvastatin in rats in vivo as a basis for clinical development of formulations for patients < 6 years.
Rosuvastatin pharmacokinetics was examined in rats aged 1, 4, 7, 10, 14, 21, 28, 35 and 42 days (from birth to sexual maturity). After intraperitoneal dose of 5 mg/kg, blood samples to determine serum rosuvastatin levels were taken at 0.5, 3 and 5 hours. Pharmacokinetic parameters (Vd, CL, AUClast, AUC0-∞) were calculated using pharmacokinecic simulations.
Both rosuvastatin CL and Vd started to increase systematically between 2 - 3 weeks of age, which was reflected by decreased total drug exposure. The AUC was up to 13 times higher in the age groups ≤14 days compared with the value at 42 days.
Based on interspecies scaling, a dose reduction could be a feasible way, how to develop appropriate dosing schedule and formulations for children aged 2 - 6 years. However, confirmation in clinical development studies will be needed.
Surfactin, a cyclic lipoheptapeptide produced by Bacillus subtilis, is a surface-active antimicrobial that targets the barrier function of lipid membranes. It inserts itself into the membrane, where ...it forms conductive pores. Depending on its concentration, it eventually disintegrates the membrane in a detergent-like manner. The molecular details of this activity are not yet sufficiently understood, nor are the mechanisms that the surfactin producer employs to resist its own toxic product. We have previously shown that B. subtilis modifies its membrane lipid composition upon the onset of surfactin production, mainly increasing the cardiolipin content. Here we show that the increased cardiolipin content leads to a decreased surfactin-induced leakage of liposomes reconstituted from lipids isolated from the surfactin producer. This stabilizing effect of cardiolipin is concentration-dependent. Using a propidium iodide-based cell permeabilization assay, we further confirmed that the cytoplasmic membrane of the mutant B. subtilis strain lacking cardiolipin was substantially more susceptible to the action of surfactin, even though the amount of bound surfactin was the same as in the wild-type strain. We propose that membrane remodelling; due to the increase in cardiolipin content, contributes to the surfactin tolerance of B. subtilis.
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•Rate of surfactin-induced lysis is slower in membranes of surfactin-adapted cells.•Cardiolipin stabilizes the most the liposome integrity against surfactin.•The stabilizing effect of cardiolipin is concentration-dependent and linear.•CL-depleted B. subtilis cells are prone to surfactin-induced permeabilization.
The historical relic of medicinal preparation of senna extract, over 200 years old, was analyzed using RP-HPLC connected with ESI
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-MS
n
. The conditions for extracting the compounds from the sample ...and separating them by HPLC were optimized. A broad spectrum of glycosides was identified. Both main senna anthraquinone glycosides, sennoside A and sennoside B, were not detected in the sample. Nevertheless, the found presence of other substances typical of senna allows the positive authentication of the sample. Three possible degradation products of sennosides were identified; rhein and two compounds with unresolved structure. Remarkable stability of some glycosides in the historical sample was found. Detailed ESI
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-MS
n
fragmentation mechanisms of sennoside A and B have been proposed.
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