Glycoproteomics is a challenging branch of proteomics because of the micro- and macro-heterogeneity of protein glycosylation. Hydrophilic interaction liquid chromatography (HILIC) is an advantageous alternative to reversed-phase chromatography for intact glycopeptide separation prior to their identification by mass spectrometry. Nowadays, several HILIC columns differing in used chemistries are commercially available. However, there is a lack of comparative studies assessing their performance, and thus providing guidance for the selection of an adequate stationary phase for different glycoproteomics applications. Here, we compare three HILIC columns recently developed by Advanced Chromatography Technologies (ACE)- with unfunctionalized (HILIC-A), polyhydroxy functionalized (HILIC-N), and aminopropyl functionalized (HILIC-B) silica- with a C18 reversed-phase column in the separation of human immunoglobulin G glycopeptides. HILIC-A and HILIC-B exhibit mixed-mode separation combining hydrophilic and ion-exchange interactions for analyte retention. Expectably, reversed-phase mode successfully separated clusters of immunoglobulin G1 and immunoglobulin G2 glycopeptides, which differ in amino acid sequence, but was not able to adequately separate different glycoforms of the same peptide. All ACE HILIC columns showed higher separation power for different glycoforms, and we show that each column separates a different group of glycopeptides more effectively than the others. Moreover, HILIC-A and HILIC-N columns separated the isobaric A2G1F1 glycopeptides of immunoglobulin G, and thus showed the potential for the elucidation of the structure of isomeric glycoforms. Furthermore, the possible retention mechanism for the HILIC columns is discussed on the basis of the determined chromatographic parameters.
- MeSH
- chromatografie iontoměničová metody MeSH
- chromatografie s reverzní fází metody MeSH
- glykopeptidy izolace a purifikace MeSH
- hydrofobní a hydrofilní interakce MeSH
- imunoglobulin G izolace a purifikace MeSH
- isomerie MeSH
- lidé MeSH
- proteomika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- srovnávací studie MeSH
The presented work proposes a novel analytical ICP-MS-based approach for the accurate and precise chromium speciation in biological tissues. The determination of total Cr(VI) and soluble Cr(III) species was carried out by alkaline EDTA extraction followed by their separation using ion-exchange high-performance liquid chromatography inductively coupled plasma mass spectrometry (IE-HPLC-ICP-MS). The developed method was validated according to the procedure given in the United States Food and Drug Administration guideline on the validation of bioanalytical methods. Validation parameters included limit of detection (≤ 0.03 μg g-1), limit of quantification (≤ 0.08 μg g-1), linearity (r ≥ 0.9998), intra-day and inter-day accuracy (86-110%) and precision (≤ 10%), extraction recovery (89-110%), carry-over effect and sensitivity. In addition, special attention was paid to the study of chromium species interconversion and the elimination of spectral interferences. Moreover, the validated ICP-MS method employing microwave acid digestion was used to determine the total Cr content in collected fractions. Finally, the whole ICP-MS-based methodology was applied to the analyses of two certified reference materials of hepatopancreas tissue. Obtained results indicated that the majority of chromium in biological tissues is bound to the solid residue, Cr(VI) was determined in none of the samples investigated. This is the first study focusing on soluble Cr(III), total Cr(VI), and total bound Cr species in biological tissues. It is characterized by efficient sample preparation and fast simultaneous analysis of Cr species with parallel total Cr analysis serving for chromium balance evaluation.
β-N-Acetylhexosaminidases (EC 3.2.1.52) are typical of their dual activity encompassing both N-acetylglucosamine and N-acetylgalactosamine substrates. Here we present the isolation and characterization of a selective β-N-acetylhexosaminidase from the fungal strain of Aspergillus versicolor. The enzyme was recombinantly expressed in Pichia pastoris KM71H in a high yield and purified in a single step using anion-exchange chromatography. Homologous molecular modeling of this enzyme identified crucial differences in the enzyme active site that may be responsible for its high selectivity for N-acetylglucosamine substrates compared to fungal β-N-acetylhexosaminidases from other sources. The enzyme was used in a sequential reaction together with a mutant β-N-acetylhexosaminidase from Talaromyces flavus with an enhanced synthetic capability, affording a bioactive disaccharide bearing an azido functional group. The azido function enabled an elegant multivalent presentation of this disaccharide on an aromatic carrier. The resulting model glycoconjugate is applicable as a selective ligand of galectin-3 - a biomedically attractive human lectin. These results highlight the importance of a general availability of robust and well-defined carbohydrate-active enzymes with tailored catalytic properties for biotechnological and biomedical applications.
- MeSH
- Aspergillus enzymologie MeSH
- beta-N-acetylhexosaminidasy chemie genetika izolace a purifikace metabolismus MeSH
- chromatografie iontoměničová MeSH
- disacharidy metabolismus MeSH
- exprese genu MeSH
- katalytická doména MeSH
- konformace proteinů MeSH
- molekulární modely MeSH
- Pichia genetika metabolismus MeSH
- rekombinantní proteiny chemie genetika izolace a purifikace metabolismus MeSH
- Talaromyces enzymologie MeSH
- Publikační typ
- časopisecké články MeSH
BACKGROUND: Insufficient specificity and invasiveness of currently used diagnostic methods raises the need for new markers of urological tumors. The aim of this study was to find a link between the urinary excretion of amino acids and the presence of urological tumors. MATERIALS AND METHODS: Using ion-exchange chromatography, we tested urine samples of patients with prostate cancer (n=30), urinary bladder cancer (n=28), renal cell carcinoma (n=16) and healthy volunteers (control group; n=21). RESULTS: In each category, we found a group of amino acids which differed in concentration compared to the control group. These differences were most significant in sarcosine in patients with prostate cancer; leucine, phenylalanine and arginine in those with bladder cancer; and sarcosine, glutamic acid, glycine, tyrosine and arginine in the those with renal cell carcinoma. CONCLUSION: Results of our research imply a possible connection between the occurrence of specific types of amino acids in the urine and the presence of urological tumors.
- MeSH
- aminokyseliny moč MeSH
- chromatografie iontoměničová MeSH
- diferenciální diagnóza MeSH
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- nádorové biomarkery * MeSH
- nádory ledvin diagnóza moč MeSH
- nádory močového měchýře diagnóza moč MeSH
- nádory prostaty diagnóza moč MeSH
- prostatický specifický antigen moč MeSH
- senioři nad 80 let MeSH
- senioři MeSH
- senzitivita a specificita MeSH
- staging nádorů MeSH
- urologické nádory diagnóza moč 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
The selectivity for 15 biogenic amines and amino acids shown by three capillary cation-exchange columns, IonPac CS19, CS12A and CS17 (250 × 0.4 mm ID, all from Thermo Fisher Scientific), exhibiting medium, medium low and ultra-low hydrophobicity, and either carboxylic or mixed carboxylic/phosphonic acid functional groups, was investigated. A mixed mode retention mechanism was revealed with ion-exchange, hydrophobic and hydrogen bonding interactions contributing to retention of polar organic molecules on these phases. The relative impact of these interactions was evaluated via the effect of concentration and pH of the eluent (methanesulfonic acid) on the retention of fifteen structurally similar biogenic amines and amino acids. Strong hydrogen bonding interactions were observed between the solute amino acid carboxylic groups and cation-exchange groups from the ion-exchangers. This is the first time retention data correlated with logP data has revealed clustering of the solutes in two groups, according to the presence or absence of a carboxylic acid functional group. In addition, stronger retention behaviour was found for the IonPac CS12A cation-exchanger, containing both carboxylic and phosphonic functional groups. Further assessment of the orthogonality plots of retention factors for the three stationary phases revealed that the columns exhibited different complimentary selectivity that can be utilised to achieve specific separations.
Recent advancements in particle design are common in reversed-phase liquid chromatography (RPLC), but in chiral separations their use is still sporadic in commercially available chiral stationary phases (CSPs). Due to reported lower mass transfer resistance, they might be a promising opportunity to increase efficiency and reduce time of analysis since the relatively higher mass transfer resistance term of CSPs caused by slow adsorption-desorption kinetics is the most performance-limiting factor in enantioselective chromatography. This study was dedicated to the evaluation of new support materials for tert-butylcarbamoylquinine (tBuCQN) based CSP to provide highly efficient and fast enantioseparations. As the main focus of this study, the chiral selector tBuCQN was immobilized on sub-2 μm fully porous particles (FPPs) and 2.7 μm superficially porous particles (SPPs) and their column performance in enantioseparation was evaluated in comparison to 5 μm FPPs by van Deemter and Knox analyses as well as kinetic plots using racemic Fmoc-Phe. Both new particle types outperformed the 5 μm FPP benchmark in terms of speed and efficiency, with wider pore materials (160 or 200 Å) being advantageous (over 90 or 120 Å). Basically decisive for the performance gain was the 10-times smaller mass transfer resistance. Furthermore, 2.7 μm 160 Å SPPs outperformed their fully porous sub-2 μm 120 Å counterpart (HminR = 4.64 μm vs. HminR = 8.94 μm) due to various parameters affording reduced plate height h of 1.7. Caused by the inaccessible core, separations were about 2-times faster. Packing of 2.7 μm core-shell particles provided a very homogeneous column bed, and, owing to its higher permeability, the column backpressure was much lower. It enables packing of longer columns providing theoretically separation efficiencies of up to 106 plates per m (as indicated by kinetic plots) and versatile use without the necessity of UHPLC systems. Investigating the effect of particle size reduction (FPPs: 5 μm, 3 μm, 1.7 μm; SPPs: 2.7 μm, 2 μm) and wider pores (FPPs: 120 Å, 200 Å; SPPs: 90 Å, 160 Å), a significantly reduced mass transfer resistance was the driving force for performance gain. Individual contributions of peak dispersion were deconvoluted for 5 μm FPP CSP and confirmed that slow adsorption-desorption kinetics is the most significant contribution to peak broadening in this chromatographic system.
Breast cancer is the most common and molecularly well-characterized malignant cancer in women; however, its progression to metastatic cancer remains lethal for 78% of patients within 5 years of diagnosis. Identifying novel markers in high risk patients using quantitative methods is essential to overcome genetic, inter-tumor, and intra-tumor variability, and to translate novel findings into cancer diagnosis and treatment. Using untargeted proteomics, we recently identified 13 proteins associated with some key factors of breast cancer aggressiveness: estrogen receptors, tumor grade, and lymph node status. Here we verified these findings in a set of 96 tumors using targeted proteomics based on selected reaction monitoring with mTRAQ labeling (mTRAQ-SRM). This study highlights a panel of gene products that could contribute to breast cancer aggressiveness and metastasis, and can help develop more precise breast cancer treatments.
- MeSH
- barvení a značení metody MeSH
- chromatografie iontoměničová metody MeSH
- hmotnostní spektrometrie metody MeSH
- lidé MeSH
- nádorové biomarkery analýza MeSH
- nádory prsu diagnóza patologie MeSH
- odběr biologického vzorku metody MeSH
- proteom analýza MeSH
- proteomika metody MeSH
- prsy patologie MeSH
- Check Tag
- lidé MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
In the enantiomeric separation of highly polar compounds, a traditionally challenging task for high-performance liquid chromatography, ion-exchange chiral stationary phases have found the main field of application. In this contribution, we present a series of novel anion-exchange-type chiral stationary phases for enantiomer separation of protected amino phosphonates and N-protected amino acids. Two of the prepared selectors possessed a double and triple bond within a single molecule. Thus, they were immobilized onto silica support employing either a thiol-ene (radical) or an azide-yne (copper(I)-catalyzed) click reaction. We evaluated the selectivity and the effect of immobilization proceeding either by the double bond of the Cinchona alkaloid or a triple bond of the carbamoyl moiety on the chromatographic performance of the chiral stationary phases using analytes with protecting groups of different size, flexibility, and π-acidity. The previously observed preference toward protecting groups possessing π-acidic units, which is a typical feature of Cinchona-based chiral stationary phases, was preserved. In addition, increasing the bulkiness of the selectors' carbamoyl units leads to significantly reduced retention times, while very high selectivity toward the tested analytes is retained.
The present study was performed to evaluate the antibacterial activities of an antimicrobial peptide (CSpK14) and the synergies thereof with β-lactams against vancomycin-resistant Staphylococcus aureus (VRSA) and Enterococci (VRE). Our strain was isolated from fermented food (kimchi), which is 99.79 % homologous with Bacillus amyloliquefaciens subsp. plantarum FZB42(T). CSpK14 was purified to homogeneity by diammonium sulfate precipitation, concentration, dialysis, and followed by two-stage chromatographic separation, i.e., Sepharose Cl-6B and Sephadex G-25 chromatography, and had a molar mass of ~4.6 kDa via Tricine SDS-PAGE and in situ examination. It was stable at pH 6.0-11.5 and temperature up to 80 °C. In addition, it was also stable with various metal ions, solvents, and proteases. The N-terminal amino acid sequence was H-Y-D-P-G-D-D-S-G-N-T-G and did not show any significant homology with reported peptides. However, it shows some degrees of identity with alpha-2-macroglobulin and ligand-gated channel protein from different microorganisms. CSpK14 significantly reduced the minimum inhibitory concentrations (MICs) of β-lactams and had no effect on non-β-lactams against VRSA and VRE. MICs of CSpK14/oxacillin and CSpK14/ampicillin were reduced by 8- to 64-fold and 2- to 16-fold, respectively. The time killing assay between CSpK14/oxacillin (2.29-2.37 Δlog10CFU/mL at 24 h) and CSpK14/ampicillin (2.30-2.38 Δlog10CFU/mL at 24 h) being >2-fold and fractional inhibitory concentration index ˂0.5 revealed synergy. Furthermore, the biofilms formed by VRSA and VRE were reduced completely. CSpK14 was simple to purify, had low molecular mass, was stable over a wide pH range or tested chemicals, had broad inhibitory spectrum, and possessed potent synergistic antimicrobial-antibiofilm properties. CSpK14 synergistically enhanced the efficacy of β-lactams and is therefore suitable for combination therapy.
- MeSH
- ampicilin farmakologie MeSH
- antibakteriální látky biosyntéza izolace a purifikace farmakologie MeSH
- Bacillus amyloliquefaciens klasifikace imunologie metabolismus MeSH
- biofilmy účinky léků růst a vývoj MeSH
- chromatografie iontoměničová MeSH
- enterokoky rezistentní vůči vankomycinu účinky léků růst a vývoj MeSH
- fylogeneze MeSH
- kationické antimikrobiální peptidy biosyntéza izolace a purifikace farmakologie MeSH
- kombinovaná farmakoterapie MeSH
- mikrobiální testy citlivosti MeSH
- oxacilin farmakologie MeSH
- rezistence na vankomycin účinky léků MeSH
- sekvence aminokyselin MeSH
- stabilita proteinů MeSH
- Staphylococcus aureus účinky léků růst a vývoj MeSH
- synergismus léků MeSH
- Publikační typ
- časopisecké články MeSH
We present the enantioseparation of new designer drugs from the cathinone family on structurally different chiral ion-exchange type stationary phases. A novel strong cation-exchange type chiral stationary phase was synthesized and its performance compared with previously reported ion-exchange type chiral stationary phases. The influence of structural elements of the chiral selectors on their chromatographic performance was studied and the possibilities of tuning chromatographic parameters by varying the polarity of the employed mobile phases were determined. Evidence is provided that a change in mobile phase composition strongly influences the solvation shell of the polarized and polarizable units of the selectors and analytes, as well as ionizable mobile phase additives. Furthermore, the structural features of the selectors (e.g. the size of aromatic units and their substitution pattern) are shown to play a key role in the effective formation of diastereomeric complexes with analytes. Thus, we have achieved the enantioseparation of all test analytes with a mass spectrometry-compatible mobile phase with a chiral strong cation-exchange type stationary phase.
