Rapid and Accurate Identification of Oligosaccharide Structures Using the Observational MSn Spectral Library

Mass spectrometry (MS) is considered to be unsuitable for the analysis of oligosaccharide structures which must distinguish a large variety of isomers with the same molecular weight. On the other hand, high sensitivity and high throughput of the strong points of MS, which is an indispensable tool for proteomics, are very attractive to glycomics. Structural analysis of oligosaccharides using MS has been achieved by detailed assignments of the fragment ions generated from tandem mass spectrometry (MS/MS) of the permethylated oligosaccharides. In MS/MS, ions of a particular m/z value are selected in the first stage of mass analysis. These "parent" or "precursor" ions are fragmented and the product ions resulting from the fragmentation are then analyzed in the second stage of mass analysis. Although MS/MS of permethylated oligosaccharides affords information on linkage positions such as 1-4 or 1-3, it is impossible to distinguish between anomers ( or ) and diastereomers (ex. GlcNAc or GalNAc). Additionally, permethylation requires at least 5  g of glycan sample, which is equal to more than 1,000 times the protein quantity required for protein identification in proteomics (1).

However, many examples in MS/MS or multistage tandem mass spectrometry (MSn) of native glycans (without permethylation) where different fragment ions and/or different intensities of the same fragment ions were observed for oligosaccharides with the same sequences, depending on the glycosidic linkages and branching structures, have recently been reported. MSn experiments of oligosaccharides in particular have shown that oligosaccharides may have characteristic fragment patterns. MSn is a technique that can only be performed on quadrupole ion-trap and FT-ICR instruments which allow the re-fragmentation of product ions (fragment ions from MS/MS), in which the product ions are trapped allowing another isolation and fragmentation to be performed, resulting in the MS3 spectrum. This process can be repeated a number of times, resulting in a series of MSn spectra where 'n' represents the number of times the isolation-fragmentation cycle has been carried out. Here, I introduce an example using matrix-assisted laser-desorption/ionization quadrupole ion trap time-of-flight (MALDI-QIT-TOF) MS.

The identification of oligosaccharide structures using the observational MSn spectral library is based on the assumption that all oligosaccharides have distinct signal intensity profiles on their MSn spectra (2). Rapid and accurate identification can be achieved by matching the MSn spectra of the analyte to the observational MSn spectral library developed by the accumulation of the MSn spectra of a large variety (3) of structure-defined oligosaccharides (Figure 1). This strategy for oligosaccharide analysis has been realized as shown by the recent achievements in the massive cloning of glycogenes making possible the preparation of a variety of oligosaccharides. Accurate identification of not only the linkage positions and branching structures but also of anomers and diastereomers has been made possible based on the MSn spectra acquired from 1 pmol of glycan sample. The advantage of this strategy is that the rapid and accurate identification of oligosaccharides can be easily performed by a researcher who is not familiar with the structural analysis of oligosaccharides using MALDI MS, which anyone can easily operate, without a detailed assignment of fragment ions, by consulting the MSn spectral library.
Akihiko Kameyama
(Research Center for Glycoscience, National Institute of Advanced Industrial Science and Technology (AIST) )
References (1) Zaia J: Mass spectromtery of oligosaccharides. Mass Spectrom. Rev, 23, 161-227, 2004
(2) Kameyama A, Kikuchi N, Nakaya S, Ito H, Sato T, Shikanai T, Takahashi Y, Takahashi K, Narimatsu H: A strategy for identification of oligosaccharide structures using observational multistage mass spectral library. Anal. Chem, 77, 4719-4725, 2005
(3) Ito H, Kameyama A, Sato T, Kiyohara K, Nakahara Y, Narimatsu H: Molecular-weight-tagged glycopeptide library: Efficient construction and applications. Angew. Chem. Int. Ed. Engl, 44, 4547-4549, 2005
Aug.31, 2005

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