Characterization of defined sulfated heparin-like oligosaccharides by electrospray ionization ion trap mass spectrometry

Glycosaminoglycans (GAG) as long, unbranched polysaccharides are major components of the extracellular matrix. Many studies provided additional evidence of a specific binding between mediators and sulfated GAG, at which the sulfation code-which means the number and positions of sulfate groups along the polysaccharide chain-plays an important role. GAG from natural sources are very inhomogeneous regarding their sulfation patterns and molecular weight. Additionally, there is a high risk of contamination. This results in a growing interest in the careful characterization of native GAG and the synthesis of artificial GAG. Additionally, chemically oversulfated GAG analogues show many favorable properties. However, the structural characterization of these carbohydrates by mass spectrometry remains challenging. One significant problem is the sulfate loss during the ionization, which increases with the number of sulfate residues. We used the sulfated pentasaccharide fondaparinux as model substance to optimize sample preparation and measurement conditions, compared different established desalination methods and already existing protocols for sulfated oligosaccharides, and investigated their impact on the quality of the mass spectra. After optimization of the measurement conditions, we could establish a gentle and fast protocol for the mass spectrometry characterization of (fully) sulfated, artificial GAG-like oligosaccharides with minimized sulfate loss in the positive and negative ion mode. Here, the negative ion mode was more sensitive in comparison with the positive one, and fondaparinux species with sulfate loss were not detectable under the optimized conditions in the positive ion mode.

Automated summary

Glycosaminoglycans (GAG) are major components of the extracellular matrix, with sulfation playing a key role in mediating their binding with mediators. Natural sources of GAG are heterogeneous in terms of sulfation patterns and molecular weight, leading to contamination risks, which has sparked interest in careful characterization and synthetic alternatives. Structural characterization of sulfated GAG analogues is challenging due to sulfate loss during ionization, with optimized conditions enabling the gentle and fast mass spectrometry characterization of (fully) sulfated, artificial GAG-like oligosaccharides with minimized sulfate loss.