title: Capillary Electrophoresis-Electrospray Ionization-Mass Spectrometry for Quantitative Analysis of Glycans Labeled with Multiplex Carbonyl-Reactive Tandem Mass Tags.
abstract: Recently developed carbonyl-reactive aminoxy tandem mass tag (aminoxyTMT) reagents enable multiplexed characterization and quantitative comparison of structurally complex glycans between different biological samples. Compared to some previously reported isotopic labeling strategies for glycans, the use of the aminoxyTMT method features a simple labeling procedure, excellent labeling efficiency, and reduced spectral complexity at the MS(1) level. Presence of the tertiary amine functionality in the reporter region of the aminoxyTMT labels leads to increased ionization efficiency of the labeled glycans thus improving electrospray ionization (ESI)-mass spectrometry (MS) detection sensitivity. The use of the labeling reagent also makes electrophoretic separation of the labeled neutral and acidic glycans feasible. In this work, we characterized the ESI and collision induced dissociation (CID) behavior of the aminoxyTMT-labeled neutral and sialylated glycans. For the high-mannose N-glycans and small sialylated oligosaccharides, CID fragmentation of [M + Na + H](2+) provides the most informative MS(2) spectra for both quantitative and qualitative analysis. For complex N-glycans, MS(3) of the protonated Y1(H) ion can be used for relative quantification without interference from the HexNAc fragments. Online capillary electrophoresis (CE)-ESI-MS/MS analyses of multiplexed aminoxyTMT-labeled human milk oligosaccharides (HMOs) and different types of N-glycans released from glycoprotein standards were demonstrated. Improved resolution and quantification accuracy of the labeled HMO isomers was achieved by coupling CE with traveling wave ion mobility (TWIM)-CID-MS/MS. N-Glycans released from human serum protein digests were labeled with six-plex aminoxyTMT and subjected to CE-ESI-MS/pseudo-MS(3) analysis, which demonstrated the potential utility of this glycan relative quantification platform for more complex biological samples.
authors.affiliation: †School of Pharmacy, University of Wisconsin, Madison, 777 Highland Avenue, Madison, Wisconsin 53705, United States.
authors.forename: Xuefei
authors.initials: X
authors.lastname: Zhong
authors.affiliation: ‡Department of Chemistry, University of Wisconsin, Madison, 1101 University Avenue, Madison, Wisconsin 53706, United States.
authors.forename: Zhengwei
authors.initials: Z
authors.lastname: Chen
authors.affiliation: §Thermo Scientific Pierce Protein Research, Thermo Fisher Scientific, Rockford, Illinois 61105, United States.
authors.forename: Sergei
authors.initials: S
authors.lastname: Snovida
authors.affiliation: ∥School of Chemistry and Chemical Engineering, Xiamen University, 361005, Xiamen, China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Liu
authors.affiliation: §Thermo Scientific Pierce Protein Research, Thermo Fisher Scientific, Rockford, Illinois 61105, United States.
authors.forename: John C
authors.initials: JC
authors.lastname: Rogers
authors.affiliation: †School of Pharmacy, University of Wisconsin, Madison, 777 Highland Avenue, Madison, Wisconsin 53705, United States.|‡Department of Chemistry, University of Wisconsin, Madison, 1101 University Avenue, Madison, Wisconsin 53706, United States.
authors.forename: Lingjun
authors.initials: L
authors.lastname: Li
mesh_terms: D019075:Electrophoresis, Capillary / Q000379:methods*; D011134:Polysaccharides / Q000032:analysis*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
chemical_list: D011134:Polysaccharides