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.

• Keywords
• Glycopeptide enrichment, Glycoproteomics, Hydrophilic interaction liquid chromatography, Immunoglobulin G, Solid-phase extraction,
• MeSH
• 2-Propanol MeSH
• Acetates MeSH
• Acetonitriles MeSH
• Chromatography, Liquid methods   MeSH
• Solid Phase Extraction methods   MeSH
• Formates * MeSH
• Glycopeptides * chemistry   MeSH
• Hydrophobic and Hydrophilic Interactions MeSH
• Immunoglobulin G chemistry   MeSH
• Humans MeSH
• Methanol * MeSH
• Solvents MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• 2-Propanol MeSH
• Acetates MeSH
• Acetonitriles MeSH
• Formates * MeSH
• formic acid MeSH Browser
• Glycopeptides * MeSH
• Immunoglobulin G MeSH
• Methanol * MeSH
• Solvents MeSH

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