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, 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)-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 lactosyl ceramide. Owing to the zero dead volume after LC separation, an extremely high sensitivity was achieved for the quantitative 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 (More papers follows, please watch our research!)

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.

Followings are various setups with different mass spectrometers.

A setup with Brucker AmaZone. 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-unit.

A setup with Bruker Esquire 3000 plus. Just remove a microscopy from a nanospray unit and stick a fluorescence ex/em probe in. But the position of the probe has to be well controlled.

This a sort of prototype!

A humanoid robot is holding a photo-probe just in front of a hole through which ions are taken by a mass spectrometer (Shimadzu, LC-IT-TOF).