Nature Methods
- 4, 577 - 582 (2007)
Published online: 27 May 2007; | doi:10.1038/nmeth1050
Strategy for the fine characterization of glycosyltransferase specificity using isotopomer assemblyHiromi Ito1, Akihiko Kameyama1, Takashi Sato1, Masako Sukegawa1, Hide-Ki Ishida2, 3 & Hisashi Narimatsu11
Glycogene Function Team of Research Center for Glycoscience, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Umezono, Tsukuba, Ibaraki
305-8568, Japan. 2
The Noguchi Institute, 1-8-1, Kaga, Itabashi-ku, Tokyo
173-0003, Japan. 3
Present address: Glyco Synthetic Lab., Tokyo Chemical Industry Co., Ltd. 6-15-9 Toshima, Kita-ku, Tokyo
114-0003, Japan.
Correspondence should be addressed to Hisashi Narimatsu h.narimatsu@aist.go.jp Glycosylation, which represents the most complex posttranslational modification (PTM) event during protein maturation, has a vital role in biological processes. Glycan biosynthesis is orchestrated by numerous glycosyltransferases, each displaying different selectivities for multiple reaction sites. The precise specificities of these enzymes have been difficult to study because of the lack of available substrates of defined structure and problems associated with the analyses. Moreover, the analysis of glycans is extremely difficult owing to the structural complexity of the glycan chain. Here we describe a new strategy for the fine characterization of enzyme specificity using substrate isotopomer assemblies. Because isotopomer assemblies contain a sugar residue that is position-specifically labeled with a stable isotope, we can use tandem mass spectrometry (MS/MS) to assign the structure of positional isomers generated by glycosylation. We demonstrated the analysis of substrate specificities of five  4-galactosyltransferases (4GalT-I, -II, -III, -IV and -V) using our strategy.
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