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.

• Keywords
• Capillary zone electrophoresis, Glycan separation, Glycopeptide separation, High-pressure liquid chromatography, Proteomics,
• Publication type
• Journal Article MeSH
• Review 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.

• Keywords
• glycopeptide separation, glycopeptides, glycoproteomics, haptoglobin, hemopexin, hydrophilic interaction liquid chromatography, retention time prediction, sex hormone-binding globulin,
• MeSH
• Chromatography, Liquid methods   MeSH
• Chromatography, Reverse-Phase * methods   MeSH
• Glycopeptides * chemistry   MeSH
• Glycosylation MeSH
• Hydrophobic and Hydrophilic Interactions MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Glycopeptides * 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
• Biomarkers metabolism   MeSH
• Chromatography, Liquid methods   MeSH
• Fucose metabolism   MeSH
• Glycopeptides metabolism   MeSH
• Glycoside Hydrolases * MeSH
• Glycosylation MeSH
• Liver Cirrhosis diagnosis   metabolism   MeSH
• Humans MeSH
• Polysaccharides metabolism   MeSH
• Tandem Mass Spectrometry methods   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• Biomarkers MeSH
• Fucose MeSH
• Glycopeptides MeSH
• Glycoside Hydrolases * MeSH
• Polysaccharides 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.

• Keywords
• Glycopeptides, Glycoproteomics, Hydrophilic interaction liquid chromatography, Immunoglobulin G, Mixed-mode chromatography,
• MeSH
• Chromatography, Ion Exchange methods   MeSH
• Chromatography, Reverse-Phase methods   MeSH
• Glycopeptides isolation & purification   MeSH
• Hydrophobic and Hydrophilic Interactions MeSH
• Immunoglobulin G isolation & purification   MeSH
• Isomerism MeSH
• Humans MeSH
• Proteomics MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Comparative Study MeSH
• Names of Substances
• Glycopeptides MeSH
• Immunoglobulin 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.

• Keywords
• glycopeptide separation, glycopeptides, glycoproteomics, hydrophilic interaction liquid chromatography, separation of glycopeptide isomers,
• MeSH
• Amides chemistry   MeSH
• Chromatography, Liquid instrumentation   methods   MeSH
• Glycopeptides chemistry   isolation & purification   metabolism   MeSH
• Glycosylation MeSH
• Hemopexin chemistry   isolation & purification   MeSH
• Hydrophobic and Hydrophilic Interactions MeSH
• Immunoglobulin G chemistry   isolation & purification   MeSH
• Isomerism MeSH
• Humans MeSH
• Polysaccharides chemistry   MeSH
• Temperature MeSH
• Trypsin chemistry   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Comparative Study MeSH
• Names of Substances
• Amides MeSH
• Glycopeptides MeSH
• Hemopexin MeSH
• Immunoglobulin G MeSH
• Polysaccharides 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.

• Keywords
• Glycopeptides, Hydrophilic interaction liquid chromatography, Solubility,
• MeSH
• Acetonitriles chemistry   MeSH
• Glycopeptides analysis   chemistry   isolation & purification   MeSH
• Hydrophobic and Hydrophilic Interactions MeSH
• N-Acetylneuraminic Acid chemistry   MeSH
• Polysaccharides chemistry   MeSH
• Solvents chemistry   MeSH
• Chromatography, High Pressure Liquid methods   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• acetonitrile MeSH Browser
• Acetonitriles MeSH
• Glycopeptides MeSH
• N-Acetylneuraminic Acid MeSH
• Polysaccharides MeSH
• Solvents 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.

• Keywords
• Glycoproteomics, Hemopexin, Hydrophilic interaction liquid chromatography, LC–MS/MS, Reversed phase chromatography,
• MeSH
• Chemistry Techniques, Analytical methods   standards   MeSH
• Chromatography, Liquid * MeSH
• Chromatography, Reverse-Phase * MeSH
• Glycopeptides analysis   MeSH
• Hemopexin analysis   MeSH
• Hydrophobic and Hydrophilic Interactions MeSH
• Polysaccharides chemistry   MeSH
• Tandem Mass Spectrometry MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Glycopeptides MeSH
• Hemopexin MeSH
• Polysaccharides MeSH