title: Fluorescence-monitored zero dead-volume nanoLC-microESI-QIT-TOF MS for analysis of fluorescently tagged glycosphingolipids.
abstract: An analysis of the glycan processing event is of particular importance to understand the nontemplate dependent synthetic mechanism of the multiple glycosylation reactions taking place in the Golgi apparatus in connection with the post-translational modification of biomolecules. In our efforts to address the issue, we constructed an analysis platform using nano-liquid chromatography (LC), which also worked as a spray tip, with an optical-fiber-based blue (470 nm) light emitting diode (LED)-induced fluorescence (520 nm) detector coupled with a microelectrospray ionization (ESI)-quadrupole ion trap (QIT)-time of flight (TOF) mass spectrometer (MS). This system was designed to enable both quantitative and qualitative analyses of fluorescently tagged molecules such as BODIPY-tagged lactosylceramide. Owing to the zero dead volume after LC separation, an extremely high sensitivity was achieved for the quantitative analysis (260 amol). It was also shown that a simultaneous online structural analysis based on MS could be achieved for the same quantity of analyte. To further demonstrate its potential, an enzymatic reaction of fluorescently tagged lactosylceramide using sialyltransferase was carried out, and the conversion yield was obtained on the basis of fluorescence detection. In addition, the structural details of a product, sialyl lactosylceramide, were obtained by MS and MS/MS analyses.
authors.affiliation: Japan Science and Technology Agency (JST), ERATO, 2-1 Hirosawa. Wako-shi, Saitama, 351-0198, Japan.
authors.forename: Shusaku
authors.initials: S
authors.lastname: Daikoku
authors.forename: Yasunari
authors.initials: Y
authors.lastname: Ono
authors.forename: Atsuko
authors.initials: A
authors.lastname: Ohtake
authors.forename: Yasuko
authors.initials: Y
authors.lastname: Hasegawa
authors.forename: Eiichiro
authors.initials: E
authors.lastname: Fukusaki
authors.forename: Katsuhiko
authors.initials: K
authors.lastname: Suzuki
authors.forename: Yukishige
authors.initials: Y
authors.lastname: Ito
authors.forename: Satoshi
authors.initials: S
authors.lastname: Goto
authors.forename: Osamu
authors.initials: O
authors.lastname: Kanie
mesh_terms: D015703:Antigens, CD / Q000737:chemistry; D002853:Chromatography, Liquid / Q000379:methods*; D005453:Fluorescence; D005679:G(M3) Ganglioside / Q000032:analysis / Q000737:chemistry; D006028:Glycosphingolipids / Q000032:analysis* / Q000737:chemistry; D007790:Lactosylceramides / Q000737:chemistry; D008832:Microchemistry / Q000379:methods; D012680:Sensitivity and Specificity; D012799:Sialyltransferases / Q000737:chemistry / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013997:Time Factors
chemical_list: D015703:Antigens, CD; D005679:G(M3) Ganglioside; D006028:Glycosphingolipids; D007790:Lactosylceramides; C009744:CDw17 antigen; D012799:Sialyltransferases