Analysis of the glycan processing event is of particular importance to understand the nontemplated synthetic mechanism of glycans utilizing multiple glycosyltransferases in the Golgi apparatus, which is a major part of the post-translational modification of biomolecules.
In our efforts to access the detailed analysis of the synthesis of glycans, 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 nano-electrospray ionization (ESI)–quadrupole ion trap (QIT) mass spectrometer (MS) capable of carrying out low-energy collision-induced dissociation. This system was designed to enable both quantitative and qualitative analyses of fluorescently tagged molecules such as BODIPY-tagged lactosyl ceramide. Owing to the zero dead volume after LC separation, an extremely high sensitivity was achieved for the analysis (524 amol).
It was also shown that a simultaneous online structural analysis based on MS could be achieved for the same quantity of analyte. An enzymatic reaction of fluorescently tagged lactosyl ceramide using sialyltransferase was carried out to demonstrate its potential. The conversion yield was obtained on the basis of fluorescence detection. In addition, the structural details of a product, sialyl lactosyl ceramide, were obtained by MS and MS/MS analyses.
References
Fluorescence-monitored zero dead-volume nano-LC-micro ESI-QIT-TOF MS for analysis of fluorescently tagged glycosphingolipids. S Daikoku, Y Ono, A Ohtake, Y Hasegawa, E Fukusaki, K Suzuki, Y Ito, S Goto, O Kanie, Analyst 2011, 136, 1046-1050.
A setup with Brucker Esquire 3000+. Nanospray unit was made in-house. A 3D stage that holds detector is held by another 3D stage, which hold both detector and a capillary. In this way, the positioning of the capillary that affects MS detection can be achieved independent of photo-detection unit.
