title: Analysis of Permethylated Glycan by Liquid Chromatography (LC) and Mass Spectrometry (MS).
abstract: The development of a reliable and high-throughput glycomic profiling strategy is in high demand due to the biological roles of glycans and their association with different diseases. Native analysis can be quite difficult because of the low ionization efficiency and microheterogeneity of glycans. In this chapter, the sample preparation protocols and LC-MS analysis of permethylated glycan strategies are introduced. Solid-phase permethylation is a fast, convenient, and high-yield method to stabilize sialic acid and improve glycan ionization efficiency and analysis in positive mode; this results in a more sensitive and reliable glycomic profiling strategy. Several modifications in the LC method are also mentioned in this chapter. Online purification simplifies sample preparation and reduces sample loss. Elevating the column temperature significantly improves the peak shape of permethylated glycans and results in isomeric separation. The identification and quantification of permethylated glycans can be achieved through high resolution MS and MS/MS experiments using a MRM method; both approaches are reliable, sensitive, and conducive to high-throughput glycomic studies.
authors.affiliation: Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, TX, 79409-1061, USA.
authors.forename: Shiyue
authors.initials: S
authors.lastname: Zhou
authors.affiliation: Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, TX, 79409-1061, USA.
authors.forename: Kerry M
authors.initials: KM
authors.lastname: Wooding
authors.affiliation: Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, TX, 79409-1061, USA. yehia.mechref@ttu.edu.
authors.forename: Yehia
authors.initials: Y
authors.lastname: Mechref
mesh_terms: D000818:Animals; D002853:Chromatography, Liquid / Q000191:economics / Q000379:methods*; D054794:Glycomics / Q000191:economics / Q000379:methods*; D006023:Glycoproteins / Q000737:chemistry; D057166:High-Throughput Screening Assays / Q000191:economics / Q000379:methods; D006801:Humans; D007536:Isomerism; D013058:Mass Spectrometry / Q000191:economics / Q000379:methods*; D008745:Methylation; D010084:Oxidation-Reduction; D011134:Polysaccharides / Q000737:chemistry*; D052616:Solid Phase Extraction / Q000379:methods
chemical_list: D006023:Glycoproteins; D011134:Polysaccharides
keywords: LC-MS; MRM; Online purification; Permethylation; Quantitative glycomics