Glycopeptide separation
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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.
- 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
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.
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
V práci sa prezentujú výsledky získané pri použití dvoch chirálnych stacionárnych fáz (CSF), založených na báze glykopeptidového antibiotika – teikoplanín aglykon (CHIROBIOTIK TAG) a metylovaný teikoplanín aglykon – m-TAG. Študovalo sa 21 racemických zmesí 1-metyl-2-piperidínoetylesterov 2-, 3- a 4- alkoxyfenylkarbámovej kyseliny. Skúmali sa interakcie medzi separovanými látkami a CSF, vplyv separácie študovaných enantiomérov na hodnotu rozlišovacieho faktora (Rij) pri dodržaní rovnakých chromatografických podmienok metódou vysokoúčinnej kvapalinovej chromatografie (HPLC). Na základe dosiahnutých výsledkov možno konštatovať, že pre dané typy racemátov je výhodnejšia CSF – CHIROBIOTIK TAG, ktorá neobsahuje sacharidovú časť, čím sa znižuje pravdepodobnosť nepolárnych interakcií, ktoré majú negatívny vplyv na hodnotu Rij.
This paper presents the results obtained with the use of two chiral stationary phases (CSP), based on a glycopeptide antibiotic agent – teicoplanin aglycone (CHIROBIOTIC TAG) and methylated teicoplanin aglycon m-TAG. Twenty-one racemic mixtures of 1-methyl-2-piperidinoethylesters of 2-, 3- a 4- alkoxyphenylcarbamic acid were examined. The investigation included interaction between separated substances and CSP, and the effect of separation of the enantiomers under study on the value of the resolution factor (Rij) under identical chromatographic conditions with the use of the method of high-performance liquid chromatography (HPLC). On the basis of obtained results, it is possible to report that CSP-CHIROBIOTIC TAG is more advantageous for these racemates, because it does not contain a saccharide part, with decrease the possibility of non-polar interactions which exert a negative effect on the Rij value.
Warfarin is a well-known anticoagulant agent that occurs in two enantiomers, (R)-(+)-warfarin and (S)-(-)-warfarin. A new liquid chromatography method for the determination of both enantiomers was developed, validated and applied in in vitro studies with the aim of evaluating the accumulation of (R)-warfarin and (S)-warfarin in the hepatoma HepG2 cell line. OptiMEM cell cultivation medium samples and cellular lysates were purified using Waters Oasis MAX extraction cartridges. The chiral separation of warfarin and the internal standard p-chlorowarfarin enantiomers was performed on an Astec Chirobiotic V2 column at a flow rate of 1.2mL/min. The mobile phase was composed of 31% acetonitrile, 5% of methanol and 64% of ammonium acetate buffer (10mmol/L, pH 4.1). The enantiomers were quantified using a fluorescence detector (lambda(excit)=320nm, lambda(emiss)=415nm). The limit of detection was found to be 0.121micromol/L of (S)-warfarin and 0.109micromol/L of (R)-warfarin. The range of applicability and linearity was estimated from 0.25 to 100micromol/L. The precision ranged from 1.3% to 12.2% of the relative standard deviation, and the accuracy reached acceptable values from 95.5% to 108.4%. The new bioanalytical method confirmed the same accumulation of (R)-warfarin and (S)-warfarin in the hepatoma HepG2 cell line.
- MeSH
- fluorescence MeSH
- glykopeptidy chemie MeSH
- lidé MeSH
- metoda nejmenších čtverců MeSH
- nádorové buněčné linie MeSH
- reprodukovatelnost výsledků MeSH
- senzitivita a specificita MeSH
- stereoizomerie MeSH
- vysokoúčinná kapalinová chromatografie metody MeSH
- warfarin analýza chemie izolace a purifikace MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- práce podpořená grantem MeSH
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 N-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.
- MeSH
- amidy chemie MeSH
- chromatografie kapalinová přístrojové vybavení metody MeSH
- glykopeptidy chemie izolace a purifikace metabolismus MeSH
- glykosylace MeSH
- hemopexin chemie izolace a purifikace MeSH
- hydrofobní a hydrofilní interakce MeSH
- imunoglobulin G chemie izolace a purifikace MeSH
- isomerie MeSH
- lidé MeSH
- polysacharidy chemie MeSH
- teplota MeSH
- trypsin chemie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- srovnávací studie MeSH
The N-glycosylation in pea seedling amine oxidase and lentil seedling amine oxidase was analyzed in the present work. For that purpose, the enzymes were purified as native proteins from their natural sources. An enzymatic deglycosylation of pea seedling amine oxidase by endoglycosidase H under denaturing conditions combined with its proteolytic digestion by trypsin was carried out in order to analyze both N-glycans and "trimmed" N-glycopeptides with a residual N-acetylglucosamine attached at the originally occupied N-glycosylation sites. The released N-glycans were subjected to a manual chromatographic purification followed by MALDI-TOF/TOF MS. MS and MS/MS analyses were also performed directly on peptides and N-glycopeptides generated by proteolytic digestion of the studied enzymes. Sequencing of glycopeptides by MALDI-TOF/TOF MS/MS after their separation on a RP using a microgradient chromatographic device clearly demonstrated binding of paucimannose and hybrid N-glycan structures at Asn558. Such carbohydrates have been reported to exist in many plant N-glycoproteins, e.g. in peroxidases. Although high-mannose glycan structures were identified after the enzymatic deglycosylation, they could not be assigned to a particular N-glycosylation site. The presence of unoccupied glycosylation sites in several peptides was also confirmed from MS/MS results.
- MeSH
- glykopeptidy analýza chemie izolace a purifikace MeSH
- glykosylace MeSH
- histaminasa analýza chemie metabolismus MeSH
- Lathyrus chemie enzymologie MeSH
- mannosyl-glykoprotein endo-beta-N-acetylglukosaminidasa chemie MeSH
- molekulární modely MeSH
- molekulární sekvence - údaje MeSH
- polysacharidy analýza chemie izolace a purifikace MeSH
- rostlinné proteiny analýza chemie metabolismus MeSH
- sekvence aminokyselin MeSH
- sekvenční seřazení MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice metody MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Analysis of the glycosylation of proteins is a challenge that requires orthogonal methods to achieve separation of the diverse glycoforms. A combination of reversed phase chromatography with tandem mass spectrometry (RP-LC-MS/MS) is one of the most powerful tools for glycopeptide analysis. In this work, we developed and compared RP-LC and hydrophilic interaction liquid chromatography (HILIC) in nanoscale on a chip combined with MS/MS in order to separate glycoforms of two peptides obtained from the tryptic digest of hemopexin. We observed reduction of the retention time with decreasing polarity of glycans attached to the same peptide backbone in HILIC. The opposite effect was observed for RP-LC. The presence of sialic acids prolonged the retention of glycopeptides in both chromatographic modes. The nanoHILIC method provided higher selectivity based on the composition of glycan, compared to nanoRP-LC but a lower sensitivity. The nanoHILIC method was able to partially separate linkage isomers of fucose (core and outer arm) on bi-antennary glycoform of SWPAVGDCSSALR glycopeptide, which is beneficial in the elucidation of the structure of the fucosylated glycoforms.
- MeSH
- chemické techniky analytické metody normy MeSH
- chromatografie kapalinová * MeSH
- chromatografie s reverzní fází * MeSH
- glykopeptidy analýza MeSH
- hemopexin analýza MeSH
- hydrofobní a hydrofilní interakce MeSH
- polysacharidy chemie MeSH
- tandemová hmotnostní spektrometrie MeSH
- Publikační typ
- časopisecké články MeSH
The composition of a sample solvent has a crucial impact on separations in hydrophilic interaction liquid chromatography (HILIC). In this short communication, we studied the effect of an organic modifier in the sample solvent on the solubility of different tryptic glycopeptides of hemopexin and haptoglobin proteins. The results showed that the solubility of glycopeptides in solvents with a high acetonitrile content depends on the type of attached N-glycan. We observed lower solubility in larger glycans attached to the same peptide backbone, and we demonstrated that glycopeptides containing sialic acids precipitate more readily than those without sialic acid. Therefore, the sample solvent composition in HILIC must be carefully optimized for accurate quantitative data collection and for adequate separation.
- MeSH
- acetonitrily chemie MeSH
- glykopeptidy analýza chemie izolace a purifikace MeSH
- hydrofobní a hydrofilní interakce MeSH
- kyselina N-acetylneuraminová chemie MeSH
- polysacharidy chemie MeSH
- rozpouštědla chemie MeSH
- vysokoúčinná kapalinová chromatografie metody MeSH
- Publikační typ
- časopisecké články MeSH
The coupling of columns in sub/supercritical fluid chromatography presents a great opportunity for influencing the separation efficiency and extending the selectivity of the separation system. Combinations of different types of chiral stationary phases could positively affect the enantioresolution if single ones are complementary to each other. In this work, two superficially porous particle (2.7 μm) macrocyclic glycopeptide-based columns, namely TeicoShell and NicoShell, were serially coupled and tested in sub/supercritical fluid chromatography for the first time. The influence of the column arrangement on the enantioseparation of structurally diverse biologically active compounds was examined. The obtained results showed how the column order crucially affected the enantioresolution of compounds tested, but the retention was negligibly affected in most cases. We also demonstrated that single TeicoShell and NicoShell columns are very promising towards the development of highly efficient and fast/ultrafast sub/supercritical fluid chromatography methods for structurally different chiral compounds. The optimized methods for sub-minute enantioselective separation of certain biologically important compounds were proposed.
