Investigation of a side reaction occurring during N-linked glycan labeling by cationic tags.

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.

Multi-charged labeling of oligosaccharides and N-linked glycans by hexahistidine-based tags for capillary electrophoresis-mass spectrometry analysis.

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.

Sample preparation for N-glycosylation analysis of therapeutic monoclonal antibodies by electrophoresis.

There are a considerable number of biopharmaceuticals that have been approved for clinical use in the past decade. Over half of these new generation drugs are glycoproteins, such as monoclonal antibodies or other recombinant glycoproteins, which are mostly produced in mammalian cell lines. The linked carbohydrate moieties affect not only their physicochemical properties and thermal stability but also crucial features like receptor-binding activity, circulating half-life, as well as immunogenicity.

Cationic labeling of oligosaccharides for electrophoretic preconcentration and separation with contactless conductivity detection.

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.