On‐line sheathless capillary electrophoresis/nanoelectrospray ionization‐tandem mass spectrometry for the analysis of glycosaminoglycan oligosaccharides

Abstract

A novel approach in glycosaminoglycomics, based on sheathless on-line capillary electrophoresis/nanoelectrospray ionization-quadrupole time of flight-mass spectrometry (CE/nanoESI-QTOF-MS) and tandem MS of extended chondroitin sulfate/dermatan (CS/DS) oligosaccharide chains is described. The methodology required the construction of a new sheathless CE/nanoESI-QTOF-MS configuration, its implementation and optimization for the high sensitivity analysis of CS/DS oligosaccharide mixtures from conditioned culture medium of decorin transfected human embryonic kidney (HEK) 293 cells. Under newly established sheathless on-line CE/(-)nanoESI conditions for glycosaminoglycan (GAG) ionization and MS detection, single CS/DS oligosaccharide components of extended chain length and increased sulfation degree were identified. Molecular ions corresponding to species carrying 5 and 6 negative charges could be generated for large GAG oligosaccharide species in the negative ion nanoESI-MS. The optimized on-line conditions enabled the detection of molecular ions assigned to oversulfated tetradeca-, octadeca-, and eicosasaccharide CS/DS molecules, which represent the category of largest sulfated GAG-derived oligosaccharides evidenced by CE/ESI-MS. By on-line CE/ESI tandem MS in data-dependent acquisition mode the oversulfated eicosasaccharide species could be sequenced and the localization of the additional sulfate group along the chain could be determined.

Discrimination of GalNAc (4S/6S) sulfation sites in chondroitin sulfate disaccharides by chip-based nanoelectrospray multistage mass spectrometry

“…Recently, analysis of oversulfation in chondroitin sulfate oligosaccharides, with determination of sulfate position along the CS chain was accomplished by either nanoelectrospray ionization quadrupole time-offlight, Fourier-transform ion cyclotron resonance mass spectrometry or combined capillary electrophoresis/ electrospray ionization mass spectrometry (CE/ESI-MS) [17,18]. However, none of these studies reported on the identification of sulfate position inside the monomer.…”

Analysis of oversulfation in a chondroitin sulfate oligosaccharide fraction from bovine aorta by nanoelectrospray ionization quadrupole time-of-flight and fourier-transform ion cyclotron resonance mass spectrometry

“…For complex mixtures, the spectrum might, therefore, become impossible to interpret due to the overlapping of isobaric species. For these reasons, the structural analysis of complex GAG mixtures claimed lately for the combination of MS with efficient separation techniques such as capillary electrophoresis [19,20,22,23], high-performance liquid chromatography [13,24], high-performance cation exchange [25], or size exclusion chromatography [26]. These strategies, however, include several analytical steps such as separation, MS screening, and sequencing of the GAG species in on-line, on-the-fly tandem MS [10], entailing specially designed interfaces and laborious optimization procedures.…”

Overview on advances in capillary electrophoresis‐mass spectrometry of carbohydrates: A tabulated review

“…Zamfir et al [74][75][76] have extensively studied dermatan sulphate (DS) and chondroitin sulfate (CS). In 2002 [74] they were the first to report the sequencing of long GAG chains derived from biological mixtures by using off-line CE-ESI-MS analysis.…”

“…The introduction of variable collision energies allowed the higher coverage of fragment ions rendering this tool more rapid, sensitive and precise for GAG analysis. Improvement of this technique was accomplished through a sheathless on-line CE-MS interface [76]. The continuous signal stability was assured by the CE-nano-ESI-MS instrument.…”

“…To date, the main articles regarding the CE-MS of saccharidic compounds found in literature include the analysis of (i) standard mixtures [42,[47][48][49][50][51][52]; (ii) carbohydrates in nutrition [19,[53][54][55][56][57][58][59]; (iii) glycans from glycoproteins [60][61][62][63][64][65]; (iv) carbohydrates in drug development [45,[66][67][68][69][70][71][72][73][74][75][76][77][78][79][80][81].…”