hydrophilic interaction Dotaz Zobrazit nápovědu
The chromatographic behavior of new biogenic purine nucleosides in hydrophilic interaction liquid chromatography was examined on three different stationary phases, namely bare silica, and amide- and cyclofructan-based stationary phases. The effects of buffer concentration, pH and acetonitrile-to-aqueous-part ratio in the mobile phase on retention and peak shape were assessed. The retention coefficients and peak symmetry values substantially differed with respect to analytes´ structures, stationary phase properties and mobile phase composition. The bare silica column was unsuitable for these compounds under the chromatographic conditions tested due to very broad and asymmetrical peaks. Furthermore, the cyclofructan-based stationary phase provided almost Gaussian peak shapes of all deazapurine nucleosides under most conditions tested. Therefore, the cyclofructan-based stationary phase is the most suitable choice for the chromatographic analysis of nucleosides.
- Klíčová slova
- Cyclofructan, Deazapurine nucleosides, Hydrophilic interaction liquid chromatography, Peak symmetry, Retention,
- MeSH
- chromatografie kapalinová metody MeSH
- fruktany MeSH
- hydrofobní a hydrofilní interakce MeSH
- koncentrace vodíkových iontů MeSH
- pufry MeSH
- purinové nukleosidy analýza MeSH
- ribonukleosidy analýza MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- fruktany MeSH
- pufry MeSH
- purinové nukleosidy MeSH
- ribonukleosidy MeSH
Hydrophilic interaction liquid chromatography is a separation technique suitable for the separation of moderately and highly polar compounds. Various stationary phases (SPs) for hydrophilic interaction liquid chromatography are commercially available. While the SPs based on the same type of ligand are available from different providers, they can display a distinct retention characteristics and separation selectivity. The current work is focused on characterization and comparison of the separation systems of two amide-based HPLC columns from two producers, i.e. XBridge Amide column and TSK gel Amide-80 column. Several characterization procedures (tests) were used to investigate the differences between these columns. The chromatographic behavior of selected analytes indicates that multimodal interactions are responsible for retention and separation on these columns. Multiple testing approaches were used in order to reveal subtle differences between the SPs. Both amide-based columns showed certain differences in retention, selectivity, and plate counts. Based on the tests used in this study, we conclude that the investigated columns provide a different degree of H-bonding interactions.
- Klíčová slova
- Amide-based columns, Hydrophilic interaction liquid chromato-graphy, Linear free energy relationship, Peptides, Selectivity tests,
- MeSH
- amidy izolace a purifikace MeSH
- chromatografie kapalinová přístrojové vybavení MeSH
- hydrofobní a hydrofilní interakce MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- amidy MeSH
Analysis of protein glycosylation is challenging due to micro- and macro-heterogeneity of the attached glycans. Hydrophilic interaction liquid chromatography (HILIC) is a mode of choice for separation of intact glycopeptides, which are inadequately resolved by reversed phase chromatography. In this work, we propose an easy-to-use model to predict retention time windows of glycopeptides in HILIC. We constructed this model based on the parameters derived from chromatographic separation of six differently glycosylated peptides obtained from tryptic digests of three plasma proteins: haptoglobin, hemopexin, and sex hormone-binding globulin. We calculated relative retention times of different glycoforms attached to the same peptide to the bi-antennary form and showed that the character of the peptide moiety did not significantly change the relative retention time differences between the glycoforms. To challenge the model, we assessed chromatographic behavior of fetuin glycopeptides experimentally, and their retention times all fell within the calculated retention time windows, which suggests that the retention time window prediction model in HILIC is sufficiently accurate. Relative retention time windows provide complementary information to mass spectrometric data, and we consider them useful for reliable determination of protein glycosylation in a site-specific manner.
- Klíčová slova
- glycopeptide separation, glycopeptides, glycoproteomics, haptoglobin, hemopexin, hydrophilic interaction liquid chromatography, retention time prediction, sex hormone-binding globulin,
- MeSH
- chromatografie kapalinová metody MeSH
- chromatografie s reverzní fází * metody MeSH
- glykopeptidy * chemie MeSH
- glykosylace MeSH
- hydrofobní a hydrofilní interakce MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- glykopeptidy * MeSH
The analysis of intact glycopeptides is a challenge because of the structural variety of the complex conjugates. In this work, we used separation involving hydrophilic interaction liquid chromatography using a superficially porous particle HALO® penta-HILIC column with tandem mass spectrometric detection for the analysis of N-glycopeptides of hemopexin. We tested the effect of the mobile phase composition on retention and separation of the glycopeptides. The results indicated that the retention of the glycopeptides was the combination of partitioning and adsorption processes. Under the optimized conditions, our HILIC method showed the ability to efficiently separate the glycoforms of the same peptide backbone including separation of the isobaric glycoforms. We achieved efficient separation of core and outer arm linked fucose of bi-antennary and tri-antennary glycoforms of the SWPAVGNCSSALR peptide and bi-antennary glycoform of the ALPQPQNVTSLLGCTH peptide, respectively. Moreover, we demonstrated the separation of antennary position of sialic acid linked via α2-6 linkage of the monosialylated glycopeptides. Glycopeptide isomers are often differentially associated with various biological processes. Therefore, chromatographic separation of the species without the need for an extensive sample preparation appears attractive for their identification, characterization, and reliable quantification.
- Klíčová slova
- Glycopeptides, Glycoproteomics, Hemopexin, Hydrophilic interaction liquid chromatography, LC-MS/MS,
- MeSH
- chromatografie kapalinová metody MeSH
- glykopeptidy analýza izolace a purifikace MeSH
- hemopexin chemie MeSH
- hydrofobní a hydrofilní interakce MeSH
- isomerie MeSH
- lidé MeSH
- proteomika metody MeSH
- sekvence aminokyselin MeSH
- tandemová hmotnostní spektrometrie metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- glykopeptidy MeSH
- hemopexin MeSH
Two polysaccharide stationary phases have been newly suggested for application in hydrophilic interaction chromatography (HILIC). Both columns (amylose-silica, 250 × 4.6 mm, 5 μm and cellulose-silica, 250 × 4.6 mm, 5 μm) demonstrated a satisfactory retention of polar compounds. The influence of the mobile-phase composition (acetonitrile content, pH, salt concentration) on the retention was in agreement with the HILIC concept. The phases showed a very similar behavior, typical efficiency of about 50,000 plates/m, cellulose retained test compounds somewhat more strongly. Under the experimental conditions, electrostatic (non-HILIC-type) interactions due to the dissociation of silanol groups on the silica surface did not influence the retention, noticeably. The applicability of polysaccharide stationary phases for the chromatography of polar compounds was proven by the separation of mixtures of sugars (fructose, glucose, saccharose, maltose, trehalose) or vitamins (nicotinamide, pyridoxine, riboflavin, thiamine, nicotinic acid, ascorbic acid).
- Klíčová slova
- Amylose, Cellulose, Hydrophilic interaction chromatography, Polysaccharide stationary phases,
- MeSH
- adsorpce MeSH
- amylosa chemie MeSH
- celulosa chemie MeSH
- chromatografie kapalinová přístrojové vybavení metody MeSH
- hydrofobní a hydrofilní interakce MeSH
- sacharidy chemie izolace a purifikace MeSH
- vitaminy chemie izolace a purifikace MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
- práce podpořená grantem MeSH
- Názvy látek
- amylosa MeSH
- celulosa MeSH
- sacharidy MeSH
- vitaminy MeSH
With the development of therapeutic oligonucleotides for antisense and gene therapies, the demand for analytical methods also increases. For the analysis of complex samples, for example plasma samples, where the use of mass detection is essential, hydrophilic interaction liquid chromatography is a suitable choice. The aim of the present work was to develop a method for separation and identification of the oligonucleotide impurities and metabolites by hydrophilic interaction liquid chromatography. First of all, the effects of different chromatographic conditions (e.g. pH of the aqueous part of the mobile phase, buffer concentration, column temperature) on the retention and separation of phosphorothioate oligonucleotides standards on the amide stationary phase were investigated. A set of model oligonucleotides containing a fully modified 21mer and its typical impurities (shortmers and oligonucleotides with different number of thiophosphate modifications) was used. The results showed that the concentration of the salt in the mobile phase as well as its pH, are the most influential parameters with regard to peak shape and separation. The knowledge gained was applied to the analysis of an unpurified 18mer oligonucleotides, analogues of the drug nusinersen used for the treatment of spinal muscular atrophy. The successful separation and identification of twenty-six and twenty-eight impurities was performed with the developed HILIC method. The method was applied to analysis of nusinersen metabolites of serum samples of patients treated with Spinraza.
- Klíčová slova
- Amide stationary phase, Hydrophilic interaction liquid chromatography, Impurities and metabolites, Nusinersen, Phosphorothioate oligonucleotides,
- MeSH
- antisense oligonukleotidy * MeSH
- chromatografie kapalinová metody MeSH
- fosforothioátové oligonukleotidy * MeSH
- hmotnostní spektrometrie metody MeSH
- hydrofobní a hydrofilní interakce MeSH
- indikátory a reagencie MeSH
- lidé MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- antisense oligonukleotidy * MeSH
- fosforothioátové oligonukleotidy * MeSH
- indikátory a reagencie MeSH
- nusinersen MeSH Prohlížeč
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.
- Klíčová slova
- Glycopeptides, Glycoproteomics, Hydrophilic interaction liquid chromatography, Immunoglobulin G, Mixed-mode chromatography,
- 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
- Názvy látek
- glykopeptidy MeSH
- imunoglobulin G MeSH
The sample preparation step is pivotal in glycoproteomic analysis. An effective approach in glycoprotein sample preparation involves enriching glycopeptides by solid-phase extraction (SPE) using polar stationary phases in hydrophilic interaction liquid chromatography (HILIC) mode. The aim of this work is to show how different experimental conditions influence the enrichment efficiency of glycopeptides from human immunoglobulin G (IgG) on an aminopropyl-modified SPE column. Different compositions of the elution solvent (acetonitrile, methanol, and isopropanol), along with varying concentrations of elution solvent acidifiers (formic and acetic acid), and different concentrations of acetonitrile for the conditioning and washing solvents (65%, 75%, and 85% acetonitrile) were tested to observe their effects on the glycopeptide enrichment process. Isopropanol proved less effective in enriching glycopeptides, while acetonitrile was the most efficient, with methanol in between. Higher formic acid concentrations in the elution solvent weakened the ionic interactions, particularly with sialylated glycopeptides. Substituting formic acid with acetic acid led to earlier elution of more glycopeptides. The acetonitrile concentration in conditioning and washing solutions played a key role; at 65% acetonitrile, glycopeptides were not retained on the SPE column and were detected in the flow-through fraction. Ultimately, it was proven that the enrichment method was applicable to human plasma samples, resulting in a significant decrease in the abundances of non-glycosylated peptides. To the best of our knowledge, this study represents the first systematic investigation into the impact of the mobile phase on glycopeptide enrichment using an aminopropyl-modified SPE column in HILIC mode. This study demonstrates the substantial impact of even minor variations in experimental conditions, which have not yet been considered in the literature, on SPE-HILIC glycopeptide enrichment. Consequently, meticulous optimization of these conditions is imperative to enhance the specificity and selectivity of glycoproteomic analysis, ensuring accurate and reliable quantification.
- Klíčová slova
- Glycopeptide enrichment, Glycoproteomics, Hydrophilic interaction liquid chromatography, Immunoglobulin G, Solid-phase extraction,
- MeSH
- 2-propanol MeSH
- acetáty MeSH
- acetonitrily MeSH
- chromatografie kapalinová metody MeSH
- extrakce na pevné fázi metody MeSH
- formiáty * MeSH
- glykopeptidy * chemie MeSH
- hydrofobní a hydrofilní interakce MeSH
- imunoglobulin G chemie MeSH
- lidé MeSH
- methanol * MeSH
- rozpouštědla MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- 2-propanol MeSH
- acetáty MeSH
- acetonitrily MeSH
- formiáty * MeSH
- formic acid MeSH Prohlížeč
- glykopeptidy * MeSH
- imunoglobulin G MeSH
- methanol * MeSH
- rozpouštědla MeSH
In this work, we have investigated retention of maltooligosaccharides and their fluorescent derivatives in hydrophilic interaction liquid chromatography using four different stationary phases. The non-derivatized maltooligosaccharides (maltose to maltoheptaose) and their derivatives with 2-aminobenzoic acid, 2-aminobenzamide, 2-aminopyridine and 8-aminonaphthalene-1,3,6-trisulfonic acid were analyzed on silica gel, aminopropyl silica, amide (carbamoyl-bonded silica) and ZIC-HILIC zwitterionic sulfobetain bonded phase. The partitioning of the analytes between the bulk mobile phase and adsorbed water-rich layer, polar and ionic interactions of analytes with stationary phase have been evaluated and compared. The effects of the mobile phase additives (0.1% (v/v) of acetic acid and ammonium acetate in concentration range 5-30 mmol L(-1)) on retention were described. The suitability of different models for prediction of retention was tested including linear solvent strength model, quadratic model, mixed-mode model, and empirical Neue-Kuss model. The mixed-mode model was extended to the parameter describing the contribution of monomeric glucose unit to the retention of non-derivatized and derivatized maltooligosaccharides, which was used for evaluation of contribution of both, oligosaccharide backbone and end-group to retention.
- Klíčová slova
- Fluorescent derivatives, Hydrophilic interaction liquid chromatography, Liquid chromatography/mass spectrometry, Oligosaccharides, Retention,
- MeSH
- 1-naftylamin chemie MeSH
- adsorpce MeSH
- aminopyridiny chemie MeSH
- chemické modely * MeSH
- chemické techniky analytické * MeSH
- chromatografie kapalinová * MeSH
- hydrofobní a hydrofilní interakce MeSH
- oligosacharidy chemie izolace a purifikace MeSH
- ortoaminobenzoáty chemie MeSH
- oxid křemičitý chemie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- 1-naftylamin MeSH
- alpha-aminopyridine MeSH Prohlížeč
- aminopyridiny MeSH
- anthranilamide MeSH Prohlížeč
- anthranilic acid MeSH Prohlížeč
- maltooligosaccharides MeSH Prohlížeč
- oligosacharidy MeSH
- ortoaminobenzoáty MeSH
- oxid křemičitý MeSH
In this work, we have investigated the predictive properties of mixed-mode retention model and oligomeric mixed-mode model, taking into account the contribution of monomeric units to the retention, in hydrophilic interaction liquid chromatography. The gradient retention times of native maltooligosaccharides and their fluorescent derivatives were predicted in the oligomeric series with number of monomeric glucose units in the range from two to seven. The maltooligosaccharides were separated on a packed column with carbamoyl-bonded silica stationary phase and 15 gradient profiles with different initial and final mobile phase composition were used with the gradient times 5; 7.5 and 10min. The predicted gradient retention times were compared for calculations based on isocratic retention data and gradient retention data, which provided better accuracy of the results. By comparing two different mobile phase additives, the more accurate retention times were predicted in mobile phases containing ammonium acetate. The acidic derivatives, prepared by reaction of an oligosaccharide with 2-aminobenzoic acid or 8-aminonaphthalene-1,3,6-trisulfonic acid, provided more accurate predictions of the retention data in comparison to native oligosaccharides or their neutral derivatives. The oligomeric mixed-mode model allowed prediction of gradient retention times using only one gradient profile, which significantly speeded-up the method development.
- Klíčová slova
- Gradient elution, Hydrophilic interaction liquid chromatography, Mixed-mode retention model, Oligomers, Prediction,
- MeSH
- chromatografie kapalinová metody MeSH
- fluorescenční barviva chemie MeSH
- hydrofobní a hydrofilní interakce MeSH
- naftaleny chemie MeSH
- oligosacharidy chemie izolace a purifikace MeSH
- ortoaminobenzoáty chemie MeSH
- oxid křemičitý MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- 8-amino-1,3,6-naphthalenetrisulfonic acid MeSH Prohlížeč
- anthranilic acid MeSH Prohlížeč
- fluorescenční barviva MeSH
- maltooligosaccharides MeSH Prohlížeč
- naftaleny MeSH
- oligosacharidy MeSH
- ortoaminobenzoáty MeSH
- oxid křemičitý MeSH