Negative-ion MALDI-QIT-TOFMSn for structural determination of fucosylated and sialylated oligosaccharides labeled with a pyrene derivative

Abstract

Oligosaccharides have many isomers and MALDI-QIT-TOFMS(n) analysis is effective for determining their structures. However, it is difficult to elucidate in detail the structures of fucosylated and/or sialylated oligosaccharides that are known to be disease markers because fucose and sialic acid residues are easily released. We have introduced a technique of labeling oligosaccharides with a pyrene derivative prior to negative-ion MALDI-QIT-TOFMS(n), and we have established a reliable method using this technique for the analysis of neutral oligosaccharides, such as fucosylated oligosaccharides containing blood group antigens H, Le(a), and Le(x). Intense and stable ionization in both positive and negative modes was achieved by derivatization with pyrene. As little as 10 fmol of pyrene-labeled oligosaccharides gave sufficient signals for analysis. Specific A-, D- or Y-type ions that depend on the structures of branching antennae could be detected by MS(n) and were useful for rapid and easy structural determination. These specific fragmentations resulting from collision-induced dissociation can be used to elucidate the structures of unknown oligosaccharides even if authentic oligosaccharides are not available as standards. By using this method, we identified and quantitated isomeric oligosaccharides with different fucosyl linkages from their mixtures. Moreover, sialylated oligosaccharide was converted to the corresponding neutral oligosaccharide by amidation, and the negative-ion spectrum was shown to be more informative than that of the original acidic oligosaccharide. Structural determination of both fucosylated and sialylated isomers, such as sialylfucosyllacto-N-hexaose I and monosialyl monofucosyllacto-N-neohexaose, was successful because fragment ions bearing fucose or amidated sialic acid were obtained on negative-MS(n).

Recent Advancement of Chemical Derivatization and Its Applications to High Sensitive Analysis of Peptide in Mass Spectrometry

“…The total number of identified N-linked glycans could be increased by about 10% [48] . Amano and coworkers reported the derivatization of oligosaccharides with pyrene butanoic acid hydrazide (PBH); as little as 10 fmol of oligosaccharides were sufficiently detected by negative-ion MALDI-TOF-MS n [49] . By the further derivatization of neutral oligosaccharide fraction from milk of a woman, total 22 oligosaccharides were identified, among which 21 had novel structures [50] .…”

Sensitive and Structure-Informative <i>N</i>-Glycosylation Analysis by MALDI-MS; Ionization, Fragmentation, and Derivatization

“…For example, fucose residues are preferentially detached from the glycan chain during CID and therefore cannot be localized by positive-ion CID. 17) Moreover, monosaccharide rearrangements may occur in the dissociation of (protonated) glycoconjugates, making MS/MS-based structural elucidation more di cult. 18,19) Recent studies have revealed that the use of the negative-ion mode in the analysis of glycoconjugates o ers distinct advantages with respect to structural elucidation.…”

Negative-ion MALDI-MS2 for discrimination of α2,3- and α2,6-sialylation on glycopeptides labeled with a pyrene derivative

“…The structural determination of glycans based on MS n has also been performed in positive-ion mode because glycans usually ionize as [M+Na] + or [M+K] + . Recently, CID of negatively charged ions has proved to be effective for the structural determination of glycans [26][27][28][29][30][31][32][33].…”

“…In particular, we have focused on the analysis of both neutral and acidic glycans in negative-ion mode, rather than positive-ion mode, because negative-ion CID of glycans derivatized with pyrene generate unique product ions for structural determination [29,30]. In general, negative-ion formation from neutral glycan is difficult, showing no or low ion yields.…”

“…In general, negative-ion formation from neutral glycan is difficult, showing no or low ion yields. By contrast, we found that glycans derivatized with pyrene produced an abundance of ions compared with other derivatization protocols such as 2-aminopyridine [29]. In order to obtain higher yields of negative ions, on-plate pyrene derivatization for glycans has been developed as a simple and rapid method [34].…”