Mass Spectrometric Fragmentation Analysis of Oligosialic and Polysialic Acids

Abstract

Oligosialic and polysialic acids (oligo/polySia) are characterized by high structural diversity, because of different types of sialic acids and glycosidic linkages. Although several methods have been described for the analysis of oligo/polySia, only high-performance liquid chromatography (HPLC) analysis in conjunction with 1,2-diamino-4,5-methylenedioxybenzene labeling, fluorometric C7/C9 detection, Western blotting, and matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy (MALDI-TOF-MS) of lactonized oligo/polySia species, require submicrogram amounts of analyte. Since these methods do not provide detailed structural information, this study is focused on the characterization of oligo/polySia by tandem mass spectrometry (MS/MS). MALDI-TOF-MS/MS and electrospray ionization tandem mass spectrometry (ESI-MS/MS), employing up to three cycles of ion isolation and fragmentation in an ion trap, have been used for the characterization of nonderivatized glycans, oligoSia species modified at their reducing or nonreducing ends, as well as partially O-acetylated oligoSia derivatives. The obtained spectra were dominated by simultaneous cleavage of glycosidic linkages and the corresponding lactone ring, whereas classical cross-ring fragments were of minor abundance. However, the combined use of the two different types of fragmentation analysis allowed a sensitive and detailed characterization of both short-chained oligoSia and long polySia species. Furthermore, oxidation of the nonreducing end sugar moiety enabled sequence determination and localization of acetylated and nonacetylated sialic acid residues.

Recent advances in sialic acid-focused glycomics

“…Kakehi et al employed LC-ESI MS after derivatization with DMB examined specific distribution of SAs in animal tissues [48]. Geyer et al used MALDI-TOF MS combined with DMB derivatization and HPLC separation method analyzed mass spectrometric fragmentation of oligosialic and polysialic acids [49]. The same group later reported a method of quantification of nucleotide-activated SAs by LC-ESI MS combined DMB derivatization and HPLC separation [50].…”

“…So far, several methods have been reported for the characterization of oligosialic and polysialic acids, among which, derivatizationwith 1,2-diamino-4,5-methylenedioxybenzene (DMB) followed by HPLC and fluorometric detection. MALDI-TOF MS has been employed as an ultrasensitive method for the characterization of oligosialic and polysialic acids [49]. In addition, lactone formation was reported by Galuska et al [51] to stabilize polysialic acids for their analysis by MALDI-TOF MS. Lactonization was achieved with TFA and o-phosphoric acid and, of several matrices tested, 6-aza-2-thiothymine (ATT) proved to be the most satisfactory with DHB being nearly as good.…”

“…Good signal to noise ratios was obtained with masses as high as 10,000 Da and polymers with up to 100 residues could be examined. Geyer et al used MALDI-TOF MS combined with lactone derivatization analyzed mass spectrometric fragmentation of oligosialic and polysialic acids [49]. Overall, MALDI-TOF MS/MS and ESI-IT-MS n are universal techniques that are suitable for detailed structural characterization of oligoSia.…”

Sialyldisaccharide conformations: a molecular dynamics perspective

“…The three dimensional structure of polysialic acids is influenced by many factors including inter-residue interactions and lactonization. It has been reported that the negative charge of the carboxylic acid at C-1 position of Neu5Ac is crucial in determining the conformation of polysialic acids and polysialic acids can form helical structure when the chain consists of more than ten monomers whereas at shorter lengths it occurs as linear chain [26,[48][49][50][51][52]. A three dimensional structural study of a(2-8) linked polysialic [53].…”

Exploration of the Sialic Acid World

“…611 For a series of papers, specifically focused on mass spectrometry of intact sialoglycans, including information about the stabilization of sialic acid, see refs. [612][613][614][615][616][617][618]. For the differentiation between (α2 !…”