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

Abstract

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...

Glycosaminoglycan Glycomics Using Mass Spectrometry

“…ESI MS-ESI MS results in relatively low vibrational excitation of analytes during the ionization process and is capable of producing ions from highly sulfated GAG oligosaccharides with minimal losses of sulfate (48). Readers wishing to analyze GAGs or other fragile biological molecules using ESI MS are advised to adjust the mass spectrometer source and ion transfer optics appropriately.…”

Electron detachment dissociation of glycosaminoglycan tetrasaccharides

“…However, the application of MS/MS to sulfated GAGs has been hindered by their large size, sulfation heterogeneity, and the lability of the sulfate groups, particularly during ion activation [10]. GAGs have been ionized by fast atom bombardment (FAB) [11,12], electrospray ionization (ESI) [13], and matrix assisted laser desorption/ionization (MALDI) [14 -17].…”

Wavelength-tunable ultraviolet photodissociation (UVPD) of heparin-derived disaccharides in a linear ion trap

“…Sulfate groups often impede access to exhaustive structural information by collision induced dissociation (CID)-MS/MS. Indeed, when the charge state of the selected molecular ion is low, neutral SO 3 losses most often predominate in comparison with glycosidic backbone cleavages [2]. This situation can be improved by combining MS/MS data resulting from different charge states.…”