Tandem mass spectrometry is a particularly powerful technique for the determination of partial oligosaccharide and glycoconjugate structural features. Multiple stages of fragmentation and analysis (MSn) show promise to extend the structural detail available. The investigators propose to develop FTICR-MS, due to its capabilities for MSn, as a """"""""carbohydrate sequenator."""""""" Precursor ions, generated by either MALDI or ESI, will be fragmented in a sequential manner to yield small (tetrasaccharides or smaller) fragments whose structure will be obtained by matching with a library of saccharide spectra (to be generated earlier as part of this work). The sequential nature of the fragment ion generation will permit the complete structure to be obtained by ordered reconstruction of the pieces. Conventional two-stage MS/MS of carbohydrates suffers from a lack of sufficient useful fragmentation for larger oligosaccharide and glycoconjugate structures. This limitation is due to the channeling of ion current into the lowest energy fragmentation pathways out of the large number of possible pathways accessible to these molecules. These fragments, observed by the investigators in numerous low-energy CID studies, are primarily glycosidic bond cleavages, with little cross-ring cleavage to yield linkage information. Furthermore, the preponderance of cleavages at the reducing side of HexNAc and NeuAc residues frequently limits the extent to which other glycosidic bond cleavages are observed. These effects serve to make tandem mass spectrometry of little use for the determination of glycosidic linkages. The investigators propose the purchase of a commercial FTICR instrument to be used for these studies. Initially, ions will be generated by microelectrospray as the alkali metal adducts. These ions will be cooled by quadrupolar excitation, ions for study will be selected by ejecting unwanted ions by SWIFT excitation, and fragmentation will be induced by SORI-CID or IR-PID. The fragments will be mass analyzed. Due to the non-destructive nature of FTICR detection, these fragment ions can be remeasured for increased S/N, or further stages of selection/excitation/detection can be performed on the same ion population.
The aim i s to generate fragmentation spectra for small pieces of the larger parent oligosaccharide. The spectra of these pieces will be matched with a library of standard spectra to determine the substructure. This library will be generated from known structures in the initial part of this work.
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