Mass spectrometry (MS) of glycoproteins is an emerging field in proteomics, poised to meet the technical demand for elucidation of the structural complexity and functions of the oligosaccharide components of molecules. Considering the divergence of the mass spectrometric methods employed for oligosaccharide analysis in recent publications, it is necessary to establish technical standards and demonstrate capabilities. In the present study of the Human Proteome Organisation (HUPO) Human Disease Glycomics/Proteome Initiative (HGPI), the same samples of transferrin and immunoglobulin-G were analyzed for N-linked oligosaccharides and their relative abundances in 20 laboratories, and the chromatographic and mass spectrometric analysis results were evaluated. In general, matrix-assisted laser desorption/ionization (MALDI) time-of-flight MS of permethylated oligosaccharide mixtures carried out in six laboratories yielded good quantitation, and the results can be correlated to those of chromatography of reductive amination derivatives. For underivatized oligosaccharide alditols, graphitized carbon-liquid chromatography (LC)/electrospray ionization (ESI) MS detecting deprotonated molecules in the negative ion mode provided acceptable quantitation. The variance of the results among these three methods was small. Detailed analyses of tryptic glycopeptides employing either nano LC/ESI MS/MS or MALDI MS demonstrated excellent capability to determine site-specific or subclass-specific glycan profiles in these samples. Taking into account the variety of MS technologies and options for distinct protocols used in this study, the results of this multi-institutional study indicate that MS-based analysis appears as the efficient method for identification and quantitation of oligosaccharides in glycomic studies and endorse the power of MS for glycopeptide characterization with high sensitivity in proteomic programs.

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A miniaturized approach was developed for quantitative permethylation of oligosaccharides, which involves packing of sodium hydroxide powder in microspin columns or fused-silica capillaries (500 μm i.d.), permitting effective derivatization in less than a minute at microscale. Prior to mass spectrometry, analytes are mixed with methyl iodide in dimethyl sulfoxide solution containing traces of water before infusing through the microreactors. This procedure minimizes oxidative degradation and peeling reactions and avoids the need of excessive clean-up. Picomole amounts of linear and branched, sialylated and neutral glycan samples were rapidly and efficiently permethylated by this approach and analyzed by matrix-assisted laser desorption/ionization mass spectrometry.Whereas structural aspects of oligosaccharides have been studied by mass spectrometry (MS) for many years, the development of matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) MS for this class of compounds has accelerated substantially the acceptance of MS-based methodologies during the last decade. In the structural analysis of complex glycans originating from various isolated glycoproteins, 1-12 MALDI-MS in conjunction with exoglycosidase digestion and a tandem (MS/MS) operation have become particularly popular. 13Although MALDI-MS structural analysis of most glycans can basically be performed in their native forms, there are several reasons for conversion of such compounds into their methylated derivatives. These include an easy determination of branching, interglycosidic linkages and the presence of configurational and conformational isomers. Permethylation also stabilizes the sialic acid residues in acidic oligosaccharides, yielding more predictable ion products when subjected to MS/MS experiments. Moreover, methylated sugars deem to ionize more efficiently than their native counterparts. In conjunction with ESI and collision-induced dissociation (CID), permethylated sugars also yield a most detailed structural information. 14-20Most permethylation procedures, employed over a number of years in carbohydrate analysis, are derived from two successful methodologies. The first, originally described by Hakomori, 21 utilizes the dimethyl sulfoxide anion (DMSO − , commonly refereed to as 'dimsyl anion') to remove protons from the sample analyte molecules prior to their replacement with methyl groups. The second, and currently more widespread approach, introduced in 1984 by Ciucanu and Kerek, 22 is based on the addition of methyl iodide to DMSO containing powdered sodium hydroxide (NaOH). The original procedure was modified more recently. 23 The current popularity of the modified procedure stems from its rapidity, experimental simplicity, 'cleaner' reaction products, 24 and the effectiveness for replacing protons at both oxygen and nitrogen sites in oligosaccharides. This methylation procedure has now been used for derivatization of Although the previously described procedures for methylation of sugars have now ...

BACKGROUND: Glycosylated proteins play important roles in cell-to-cell interactions, immunosurveillance, and a variety of receptor-mediated and specific protein functions through a highly complex repertoire of glycan structures. Aberrant glycosylation has been implicated in cancer for many years.

Glycomic profiles derived from human blood sera of 10 healthy males were compared to those from 24 prostate cancer patients. The profiles were acquired using MALDI-MS of permethylated N-glycans released from 10-microL sample aliquots. Quantitative permethylation was attained using solid-phase permethylation. Principal component analysis of the glycomic profiles revealed significant differences among the two sets, allowing their distinct clustering. The first principal component distinguished the 24 prostate cancer patients from the healthy individuals. It was determined that fucosylation of glycan structures is generally higher in cancer samples (ANOVA test p-value of 0.0006). Although more than 50 N-glycan structures were determined, 12 glycan structures, of which six were fucosylated, were significantly different between the two sample sets. Significant differences were confirmed through two independent statistical tests (ANOVA and ROC analyses). Ten of these structures had significantly higher relative intensities in the case of the cancer samples, while the other two were less abundant in the cancer samples. All 12 structures were statistically significant, as suggested by their very low ANOVA scores (<0.001) and ROC analysis, with area under the curve values close to 1 or 0. Accordingly, these structures can be considered as cancer-specific glycans and potential prostate cancer biomarkers. Therefore, serum glycomic profiling appears worthy of further investigation to define its role in cancer early detection and prognostication.