DEVELOPMENT OF AN LC/MS INTERFACE FOR ANALYSIS OF GLYCANS IN THE NEGATIVE MODE
One of the primary factors limiting progress in profiling of native and reductively aminated glycan classes has been the stability of the HPLC-MS spray interface. Native glycans are best analyzed using negative polarity, and our experience has been that conventional silica sprayers do not deliver ion signal stable enough to allow analysis of large sample sets. Such analyses requires continuous operation over the course of a week or more. We have implemented two LC/MS interfaces that solve this problem. As part of the most recent P41 renewal, in summer, 2007 we installed a Waters NanoAcquity UPLC system with the Thermo-Finnigan LTQ-Orbitrap system. This high pressure chromatography system has the advantage that it delivers un-split flow through capillary columns, decreasing problems associated with particulates that slowly accumulate in the spray interface in the negative mode. In fall, 2007, we installed the Advion Triversa Nanomate robot funded through the most recent P41 renewal. This system increased the spray stability in both on-line and off-line LC/MS modes. Both positive ion proteomics and negative ion glycomics projects now use the Advion robot as the spray interface for all on-line and off-line analyses.
An Agilent 6520 QTOF mass spectrometer with chip-based chromatography system was installed winter, 2008. As part of a collaboration with Agilent, we demonstrated an amide-silica hydrophilic interaction chromatography system for glycan analysis. The chip-based system incorporates a 75 micron x 15 cm column, trapping cartridge and sprayer. The robotic interface removes any need for optimization of spray geometry or tubing connections. As a result, we are now able to generate glycomics datasets using a robust spray interface so as to achieve reproducible instrumental response over time.
