Efficient recovery of glycosaminoglycan oligosaccharides from polyacrylamide gel electrophoresis combined with mass spectrometry analysis

Abstract

To promote efficient separation and structural analysis of glycosaminoglycan oligosaccharides, we developed a straightforward method that combined gel electrophoresis and mass spectrometry (MS). Potential limitations of this approach (e.g., low extraction yields and weak compatibility with MS) were resolved by developing an active extraction procedure that yielded a quantitative amount of sulfated oligosaccharides from excised gel bands. The compatibility of obtained oligosaccharides for subsequent MS analysis was ensured using a single, simple clean-up step on a mixed C18/graphite carbon solid-phase column that was fully effective for polymerization degrees ranging from di- to dodecasaccharides. The reported combination of carbohydrates-polyacrylamide gel electrophoresis with MS was successfully applied to glucosamino- (heparin) and galactosamino- (dermantan sulfate) glycans, demonstrating the potential of our method for structural analysis of bioactive sulfated carbohydrates extracted from biological matrices. Graphical Abstract ᅟ.

Investigation of action pattern of a novel chondroitin sulfate/dermatan sulfate 4-O-endosulfatase

“…Desulfation of synthetic CS oligosaccharides by 4-O-endosulfatase was carried out as previously described (28). Briefly, 10 μl of the enzyme preparation was mixed with 5 μL of CS oligosaccharides (100 μM final) in 35 μL of 72 mM phosphate buffer, pH 8.1 (50 mM final), and incubated at 30 °C, for 1, 2, 3, 4, 5, 6 and 24 h. After incubation, the resulting oligosaccharides were isolated and desalted using a C18 + Carbon-SPE TopTip cartridge (Glygen, Columbia, MD, USA) as previously described (36). Each elution fraction was pooled, freeze-dried, and dissolved in 10 μl water for MS analysis.…”

Analysis of the Glycosaminoglycan Chains of Proteoglycans

“…The GAG fractions obtained from PAGE can often be directly applied to MS analysis for sequencing. 16,19…”

Farklı çapraz bağlayıcılar ile hazırlanmış poliakrilamid hidrojellerinin değerlendirilmesi

“…Poliakrilamid; hidrojel tanımlaması yapıldığı günden beri üzerinde en çok çalışılan hidrojel olup, günümüzde absorban ve toprak düzenleyici olarak, atık suların arıtılmasında, kozmetik, kağıt ve tekstil endüstrisinde, elektroforez uygulamalarında yaygın kullanıma sahiptir [1][2][3][4][5][6][7]. Vinilik özellikteki sade monomeri çok kolay polimerleşir ve bu özelliği en çok tercih edilme nedenlerinden birisidir.…”