AN LC/MS PLATFORM FOR REDUCTIVE AMINATION-TAGGED GLYCANS

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Mass spectrometric glycan quantitation is facilitated by the use of stable isotope glycan tags through reductive amination chemistry. Such tags may be used with released glycans containing an aldehyde reducing end. A tetraplex tag has been developed at the MSR and tested using N-glycans and glycosaminoglycans. Both glycan classes may be separated using amide-hydrophilic interaction chromatography (HILIC) using mass spectrometry compatible mobile phases. In order to make quantitative comparisons of glycan expression, it is necessary to develop a platform that allows robust and reproducible data acquisition and facile data reduction. As part of this effort, the MSR is collaborating with Agilent Technologies Corp. to test a chip-based spray interface for glycan LC/MS. The chip sprayer was packed with amide-silica stationary phase and tested using N-glycans, heparin, heparan sulfates, and chondroitin sulfates. The results showed that very robust and stable spray could be easily obtained for all glycan classes tested. The glycans tended to produce sample carry over in initial results. This problem should be solvable with more extensive washing of the autosampler needle. The data consist of mass chromatograms in which a given glycan composition is observed in 2-3 charge states. The lowest charge state is typically observed with ammonium adducts. Thus, each composition is observed as 4-5 m/z values. A typical mixture consistes of several dozen compositions, giving rise to very complex spectral patterns. It is objective of this collaboration to develop software routines for extracting, de-convoluting and combining all LC/MS ions so as to reduce the data into readily interpretable glycan composition and abundance tables.