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.

Permethylation of glycans prior to their mass spectrometric determination has now become a time-honored methodology in glycoconjugate analysis due to the advantage of a simultaneous analysis of neutral and acidic glycans as well as enhanced sensitivity and easier tandem mass spectrometry interpretation. While the different solvent extraction-based versions of this method often suffice in different structural studies, they are generally less satisfactory in the quantitative determinations aiming at minor quantities of the analyzed materials. To overcome these difficulties, we recently introduced a solid-phase capillary permethylation technique (Kang et al., Rapid Commun. Mass Spectrom. 2005; 19: 3421) for microscale determination. Here, we describe a very useful high-throughput extension of the solid-phase methodology utilizing spin columns packed with sodium hydroxide beads. This procedure has been thoroughly optimized to match the analytical performance parameters of the previously used capillary technique. As demonstrated with a high-precision glycomic profiling analysis of human blood serum, this methodological improvement offers simplicity and reproducibility, allowing the complete permethylation of 12-18 samples in less than 20 min.

Comparative glycan quantification has thus far been a challenging task due to the lack of sensitive and reproducible analytical techniques. We introduce here a combination of quantitative permethylation and isotope labeling of glycans as an approach (C-GlycoMAP) allowing precise comparison between different samples in a single MALDI-MS analysis. Samples are either methylated or deuteriomethylated prior to their mixing and mass spectrometric acquisitions. Comparative analyses are based on the ratio of the two isotopically distinct forms of the same glycan structure, thus allowing a direct absolute evaluation of the intensities of the two forms originating from two different biological samples (e.g., control and diseased). The direct comparison between the two forms eliminates a MALDI-MS low m/z bias commonly associated with this technique. These comparative analyses are highly reliable when the intensity ratios of the two forms lie between 0.125 and 6, an overall reproducibility better than 30% (RSD). The value of C-GlycoMAP is demonstrated here for N-glycans derived from human blood serum collected from a healthy individual and a breast cancer patient as well as for O-glycans derived from normal and cancer cell extracts.

This chapter discusses in detail a miniaturized version of the widely used permethylation technique which permits quantitative derivatization of oligosaccharides derived from minute quantities of glycoprotein. The approach involves packing of sodium hydroxide powder or beads in a microcolumn format, including spin columns, fused silica capillaries (500 microm i.d.) and plastic tubes (1 mm i.d.). The derivatization proceeds effectively in less than a minute time scale and it is applicable to glycans derived from femtomole quantities of glycoproteins. Prior to mass spectrometry (MS), methyl iodide is added to analytes suspended in dimethyl sulfoxide solution containing traces of water. The reaction mixture is then immediately infused through the microreactor. The packed sodium hydroxide powder or beads inside the microcolumns minimize oxidative degradation and peeling reactions which are otherwise commonly associated with the conventional permethylation technique. In addition, this solid-phase permethylation approach eliminates the need for excessive sample clean-up. As demonstrated below, picomole amounts of various types of glycans derived from model glycoproteins as well as real samples, including linear and branched, sialylated and neutral glycans were shown to become rapidly and efficiently permethylated through this approach.