Capillary electrophoresis-mass spectrometry was applied for the analysis of oligosaccharides and N-linked glycans with an attached charge label facilitating electrophoretic migration and electrospray ionization efficiency. Several different labeling strategies have been tested with different tags and tagging reactions including reductive amination and hydrazone formation. However, a formation of multiple labeled N-linked glycans was observed by CE-MS in a positive ion mode when positively charged labels such as aliphatic amines containing a quaternary ammonium group were attached to N-linked glycans by reductive amination. A reaction mechanism explaining a side reaction occurring during the labeling and the multiple product formation was proposed and confirmed by using isotopically labeled N-acetylglucosamine. Finally, it was confirmed that derivatization of sugars via a hydrazone formation can be a simpler method with a high reaction yield suitable for high sensitive CE-ESI/MS analyses of N-linked glycans.
The labeling by amino acids and peptides was investigated for sensitive and fast analyses of oligosaccharides and N-linked glycans by capillary electrophoresis-mass spectrometry (CE-MS). Peptide tags with a various number of histidine residues were tested for maltooligosaccharide labeling in order to investigate the effect of the size of labels and a number of charges on CE-MS analysis. Nevertheless, the reductive amination labeling of N-linked glycans by a hexahistidine tag resulted in a multiple products formation, therefore a peptide tag was modified by hydrazine functionality in order to perform labeling by hydrazone formation chemistry. This labeling approach significantly improved sensitivity with LOD of labeled maltopentaose determined to be 40 nmol/L and also significantly reduced separation time of neutral maltooligosaccharides and N-linked glycans released from bovine ribonuclease B. Furthermore, the labeling by this multi-cationic peptide hydrazine tag also allowed performing analysis of acidic glycans by CE-MS in a positive ion mode as demonstrated by separation of sialylated N-linked glycans released from bovine fetuin.
- MeSH
- elektroforéza kapilární metody MeSH
- fetuin A chemie MeSH
- histidin chemie MeSH
- hmotnostní spektrometrie metody MeSH
- oligopeptidy chemie MeSH
- oligosacharidy analýza chemie izolace a purifikace MeSH
- polysacharidy analýza chemie izolace a purifikace MeSH
- ribonukleasy chemie MeSH
- skot MeSH
- zvířata MeSH
- Check Tag
- skot MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Cationic derivatization of oligosaccharides by quaternary ammonium label was investigated for capillary electrophoretic separation with transient isotachophoretic preconcentration (t-ITP) as detected by capacitively coupled contactless conductivity detection (C4D). Dextran ladder, prepared by partial hydrolysis of dextran, isomaltotriose, maltopentaose and maltoheptaose were derivatized by reductive amination with (2-aminoethyl)trimethylammonium chloride. This label provides permanent positive charge potentially also useful for mass spectrometry ionization. Capillary electrophoresis (CE) separations of the oligosaccharides were tested in zone electrophoretic (CZE) and combined t-ITP/CZE modes. The transient isotachophoretic preconcentration was performed by injecting the sample solution complemented by ammonium acetate into the acetic acid background electrolyte. In this case the ammonium ions served as leading electrolyte followed by the sample zone and the background electrolyte of acetic acid acted as terminating electrolyte. The oligosaccharides were focused into narrow zones by ITP principles and during the course of migration through the separation capillary relaxed into zone electrophoretic separation mode. The separated ones were detected by C4D detector with detection limits in the nanomolar concentration range.
