Summary
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 μm 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.
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Acknowledgments
This work was supported by grants No. GM24349 from the National Institute of General Medical Sciences, U.S. Department of Health and Human Services, and RR018942 from the National Institute of Health through the National Center for Research Resources (NCRR) for the National Center for Glycomics and Glycoproteomics. PK is the recipient of a fellowship from Merck Research laboratory.
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Mechref, Y., Kang, P., Novotny, M. (2009). Solid-Phase Permethylation for Glycomic Analysis. In: Packer, N.H., Karlsson, N.G. (eds) Glycomics. Methods in Molecular Biology™, vol 534. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-59745-022-5_4
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DOI: https://doi.org/10.1007/978-1-59745-022-5_4
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