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 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