Influence of Charge State on Product Ion Mass Spectra and the Determination of 4S/6S Sulfation Sequence of Chondroitin Sulfate Oligosaccharides

A combination of negative ion nano-electrospray ionization Fourier-transform ion cyclotron resonance and quadrupole time-of-flight mass spectrometry was applied to analysis of oversulfation in glycosaminoglycan oligosaccharides of the chondroitin sulfate type from bovine aorta. Taking advantage of the high-resolution and high mass accuracy provided by the FT-ICR instrument, a direct compositional assignment of all species present in the mixture can be obtained. An oligosaccharide fraction containing mainly hexasaccharides exhibited different levels of sulfation, indicated by the presence of species with regular sulfation pattern as well as oversulfated oligosaccharides with one additional sulfate group. Oversulfation can be directly identified from the high-resolution/high mass accuracy FT-ICR mass spectra according to their specific isotopic fine structure. Location of sulfate groups was analyzed by Q-TOF MS and low-energy CID MS/MS. Tetrasulfated hexasaccharides were analyzed by use of collision-induced dissociation at variable collision energy for an unambiguous assignment of the attachment site of the sulfate groups by minimizing unspecific neutral losses. Cleavage of glycosidic bonds gave rise to B-and C-type ions and their respective complementary Y-and Z-type fragment ions. (J Am Soc Mass Spectrom 2007, 18, 179 -187)

Section: Structural Characterization Of the Hexasaccharide Species Bymentioning

confidence: 99%

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confidence: 99%

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

Mormann

Zamfir

Seidler

et al. 2007

“…Although FAB tandem MS has been used to study CS oligosaccharides up to tetramers size, the singly charged nature of these ions resulted in abundant fragments resulting from loss of sulfate groups [22]. Intact molecular ions could, however, be produced using electrospray ionization (ESI) in the negative mode, and tandem MS has been applied to the CS oligosaccharide ions so generated [23,24]. Because CS disaccharides sulfated at the 4-or 6-position of the GalNAc residue, respectively, produce distinct CID product ion patterns [25][26][27], quantitative measurements based on ion abundances provide useful information on the global domain organization of CS chains [28].…”

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confidence: 99%

“…Because CS disaccharides sulfated at the 4-or 6-position of the GalNAc residue, respectively, produce distinct CID product ion patterns [25][26][27], quantitative measurements based on ion abundances provide useful information on the global domain organization of CS chains [28]. It has also been shown that the information content of tandem mass spectra generated from CS oligosaccharides is strongly dependent on the charge state of the precursor ion [24]. Further, it was shown that the relative abundances of the individual glycosidic bond cleavage ions formed reflect the position of GalNAc sulfation [23].…”

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confidence: 99%

Tandem mass spectrometric strategies for determination of sulfation positions and uronic acid epimerization in chondroitin sulfate oligosaccharides

Zaia

Chan

et al. 2003

Self Cite

102

Chondroitin sulfate (CS) is a glycosaminoglycan consisting of repeating (HexA-GalNAc sulfate) disaccharides, the functions of which depend on patterns of sulfation and uronic acid epimerization. The correlation of biological activities with structure requires a strategy to determine the sequences of CS oligosaccharides without the need for total isolation. Tandem mass spectrometry has enabled the development of proteomics, based on CID fragmentation of ions produced from complex mixtures of proteolytic peptides, and has the potential for rapid sequencing of CS and other glycosaminoglycan classes. The most challenging aspects of CS sequencing are to distinguish GalNAc residues sulfated at the 4-versus the 6-position and uronic acid epimers. This work describes the utility of (1) reducing terminal derivatives and (2) control of precursor ion charge state for tandem mass spectrometric strategies for determining GalNAc sulfation positional isomers of CS. The capability of tandem MS to differentiate uronic acid epimers is also shown, providing evidence that complete or nearly complete information on CS covalent structure may be obtained using tandem

“…Negative ESI tandem MS studies of chondroitin sulfate oligosaccharides have shown that the product ion pattern depends strongly on the charge state of the precursor ion [21,22]. Specifically, it was shown that ions for which all sulfate groups are deprotonated fragment to produce abundant glycosidic bond cleavages without losses of the sulfate groups.…”

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confidence: 99%

Competing fragmentation processes in tandem mass spectra of heparin-like glycosaminoglycans

Naggar

Costello

Zaia

2004

Self Cite

Heparin-like glycosaminoglycans (HLGAGs) are highly sulfated, linear carbohydrates attached to proteoglycan core proteins and expressed on cell surfaces and in basement membranes. These carbohydrates bind several families of growth factors and growth factor receptors and act as coreceptors for these molecules. Tandem mass spectrometry has the potential to increase our understanding of the biological significance of HLGAG expression by providing a facile means for sequencing these molecules without the need for time-consuming total purification. The challenge for tandem mass spectrometric analysis of HLGAGs is to produce abundant ions derived via glycosidic bond cleavages while minimizing the abundances of ions produced from elimination of the fragile sulfate groups. This work describes the competing fragmentation pathways that result from dissociation of high negative charge state ions generated from HLGAGs. Glycosidic bond cleavage ion formation competes with losses of equivalents of H 2 SO 4 , resulting in complex ion patterns. For the most highly sulfated structure examined, an octasulfated tetramer, an unusual loss of charge from the precursor ion was observed, accompanied by low abundance ions originating from subsequent backbone cleavages. These results demonstrate that fragmentation processes competing with glycosidic bond cleavages are more favored for highly sulfated HLGAG ions. In conclusion, reduction of charge-charge repulsions, such as is achieved by pairing the HLGAG ions with metal cations, is necessary in order to minimize the abundances of ions derived via fragmentation processes that compete with glycosidic bond cleavages. Sequence determination, including localization of sulfate groups on HLGAGs, is of significant interest, as their biological activities are believed to be expressed through patterns of sulfation and uronic acid epimerization [4,5].HLGAGs have traditionally proven difficult to analyze using mass spectrometry. Efforts to analyze these molecules using fast atom bombardment MS resulted in the observation of losses of SO 3 and NaSO 3 from the precursor ion, in addition to glycosidic bond cleavage ions [6 -10]. The poor sensitivity, relative to that achieved for peptides of comparable size, and lack of an intact precursor ion meant that tandem MS was not a viable analysis option when FAB ionization was employed. Although HLGAGs produce weak signals by matrix-assisted laser desorption/ionization (MALDI), much more abundant ions are detected from complexes between these molecules and a basic peptide [11][12][13]. In conjunction with enzymatic and/or chemical degradation, this MALDI sample preparation technique has been effectively used to sequence highly purified HLGAGs [14 -17]. Because the ions are detected in the positive mode, however, the charge is carried by the basic peptide and tandem MS of the HLGAG is not possible.Because HLGAGs and other sulfated carbohydrates produce abundant negative ions using electrospray ionization (ESI) without requiring basic peptide complexation [18...