Glycomics & Glycoproteomics


Fast Small-scale Permethylation Procedure for the Structural Characterization of Oligosaccharides by MALDI/TOF/TOF Tandem Mass Spectrometry

Mass spectrometry (MS) is routinely employed as a key methodology in the structural elucidation of glycoconjugates. Although mass spectrometers using single mass analyzers can now readily assist acquisition of compositional data on glycans in terms of isobaric monosaccharides, the structural analysis of glycans requires determination of branching, linkage position and monomer anomericity. The aim of this study has been the development of miniaturized permethylation procedure for the derivatization of glycans which are subsequently analyzed by MALDI/TOF/TOF tandem MS.

The procedure is based on a permethylation approach developed by Ciucanu and Costello (J. Am. Chem. Soc. 2003, 125, 16213); its miniaturized version involves packing of sodium hydroxide powder in microspin columns or fused silica capillaries (500 mm i.d.). This approach permits highly effective permethylation of various oligosaccharides in less than a minute. It involves the suspension of analytes and methyl iodide in dimethyl sulfoxide prior to infusing through the packed microspin or fused silica capillary columns. Moreover, this approach eliminates the need for excessive sample clean-up prior to MS analysis. Picomole levels of linear and branched sialylated and neutral glycan samples were effectively and rapidly permethylated through this approach.


Figure 2. 3-D recording of capillary LC/MALDI/TOF/TOF MS and tandem MS of permethylated, reduced N-glycans derived from ribonuclease B. Glycans were chemically released by ß-elimination, permethylated through the capillary approach, separated on a reversed-phase C18 capillary column, while capillary LC eluents were mixed with matrix, deposited on MALDI plate and analyzed by MALDI/TOF/TOF MS. Insets are the MSMS spectra of separated glycan structures.


Serial Lectin Affinity Chromatography – Mass Spectrometry: Fast and Easy Method for Analysis of Glycoproteins

Glycosylation of proteins is considered to be one of the most common and diverse post-translational modifications proteins undergo in mammalian cells. Because of a proteome complexity, analysis based on single one-dimensional LC/MS/MS is often not sufficient to account for all proteins present in a sample. Moreover, the characterization of glycosylated proteins is even more difficult because, glycopeptides are weakly ionized in the electrospray process when compared to peptides. Accordingly, additional simplification of a complex mixture is usually needed and accomplished by fractionation. Lectin affinity chromatography has been widely used for fractionation of glycoproteins. Due to the different trapping capability towards various carbohydrate structures, lectins used in series and coupled on-line to nanoLC/MS/MS, offer an interesting, fast and easy approach to resolve sample microheterogenity.