Assessing Fucosyltransferase Activity via MSn

Abstract

Fucosyltransferases add fucose residues to oligosaccharides. Resulting fucosylated epitopes include the common core fucosylation of N‐glycans, as well as important ligands such as Lewis structures and H antigens. Many of these structural motifs can be found on N‐linked glycans, O‐linked glycans, and glycosphingolipids. In complex biological samples, multiple fucosyltransferases, as well as multiple substrate specificities of individual fucosyltransferases, can result in multiple isomeric forms within individual glycan compositions. Intact mass analysis alone will not distinguish these isomers. Chromatographic separation may or may not resolve these structures, and, will not provide direct structure identification.Typically, MS/MS analysis of fucosylated N‐linked glycans can distinguish core fucosylation from antennal fucosylation. Finer details such as distinguishing Lewis structures from H antigens is a more difficult problem, as fucosylated lactosamine antennae fragments are produced from both structures. Disassembling these structural fragments to a further stage, MS3, in an ion trap, can easily distinguish Lewis from H antigen structures, providing direct empirical evidence of structure.We demonstrate MSn data from a variety of samples. Fucosyltransferase activity was assessed through transfection of fucosyltransferase (3,4,5,6,7, and 9) genes into mesenchymal stem cells, with comparison to non‐transfected cells as a control. MCF‐7 and MDA‐MB‐231 cultured cells were also interrogated for fucosylation isomers. Glycophorin C was also interrogated, with fucosylation on polylactosamine structures.For cultured cells, glycolipids were extracted via solvent extraction. Proteins were enzymatically digested, followed by enzymatic release of N‐linked glycans. Released N‐glycans were separated from O‐glycopeptides; O‐glycans were released by reductive b‐elimination. Purified glycan and glycosphingolipid fractions were permethylated. Glycan samples were directly infused into the mass spectrometer via nano‐ESI. Precursor ion selection was made manually.Fucosyltransferase‐transfected mesenchymal stem cells were interrogated for synthesis of Lewis X structures, with data shown to indicate that different fucosyltransferases show differing preferences for synthesizing Lewis X vs. sialylated Lewis X.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.