Glycosylation is one of the most significant and abundant post-translational modifications in cells. Glycomic and glycoproteomic analyses involve the characterization of oligosaccharides (glycans) conjugated to proteins. Glycomic and glycoproteomic analysis is highly challenging because of the large diversity of structures, low abundance, site-specific heterogeneity, and poor ionization efficiency of glycans and glycopeptides in mass spectrometry (MS). MS is a key tool for characterization of glycans and glycopeptides. However, MS alone does not always provide full structural and quantitative information for many reasons, and thus MS is combined with some separation technique. This review focuses on the role of separation techniques used in glycomic and glycoproteomic analyses, liquid chromatography and capillary electrophoresis. The most important separation conditions and results are presented and discussed.

• Klíčová slova
• Capillary zone electrophoresis, Glycan separation, Glycopeptide separation, High-pressure liquid chromatography, Proteomics,
• Publikační typ
• časopisecké články MeSH
• přehledy 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

Carbohydrates form one of the major groups of biological macromolecules in living organisms. Many biological processes including protein folding, stability, immune response, and receptor activation are regulated by glycosylation. Fucosylation of proteins regulates such processes and is associated with various diseases including autoimmunity and cancer. Mass spectrometry efficiently identifies structures of fucosylated glycans or sites of core fucosylated N-glycopeptides but quantification of the glycopeptides remains less explored. We performed experiments that facilitate quantitative analysis of the core fucosylation of proteins with partial structural resolution of the glycans and we present results of the mass spectrometric SWATH-type DIA analysis of relative abundances of the core fucosylated glycoforms of 45 glycopeptides to their nonfucosylated glycoforms derived from 18 serum proteins in liver disease of different etiologies. Our results show that a combination of soft fragmentation with exoglycosidases is efficient at the assignment and quantification of the core fucosylated N-glycoforms at specific sites of protein attachment. In addition, our results show that disease-associated changes in core fucosylation are peptide-dependent and further differ by branching of the core fucosylated glycans. Further studies are needed to verify whether tri- and tetra-antennary core fucosylated glycopeptides could be used as markers of liver disease progression.

• MeSH
• biologické markery metabolismus   MeSH
• chromatografie kapalinová metody   MeSH
• fukosa metabolismus   MeSH
• glykopeptidy metabolismus   MeSH
• glykosidhydrolasy * MeSH
• glykosylace MeSH
• jaterní cirhóza diagnóza   metabolismus   MeSH
• lidé MeSH
• polysacharidy metabolismus   MeSH
• tandemová hmotnostní spektrometrie metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• biologické markery MeSH
• fukosa MeSH
• glykopeptidy MeSH
• glykosidhydrolasy * MeSH
• polysacharidy MeSH

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

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.

• Klíčová slova
• glycopeptide separation, glycopeptides, glycoproteomics, hydrophilic interaction liquid chromatography, separation of glycopeptide 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
• Názvy látek
• amidy MeSH
• glykopeptidy MeSH
• hemopexin MeSH
• imunoglobulin G MeSH
• polysacharidy MeSH
• trypsin 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.

• Klíčová slova
• Glycopeptides, Hydrophilic interaction liquid chromatography, Solubility,
• 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
• Názvy látek
• acetonitrile MeSH Prohlížeč
• acetonitrily MeSH
• glykopeptidy MeSH
• kyselina N-acetylneuraminová MeSH
• polysacharidy MeSH
• rozpouštědla MeSH