This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Although ECD has proven its usefulness for proteins, peptides and carbohydrates, its applications are limited by the fact that its use is almost completely restricted to Fourier transform ion cyclotron resonance mass spectrometers. To make electron-based methods practical on instruments other than FTMS instruments, Hunt et al. introduced a new method for dissociation that relies on electron transfer by negative species (usually fluoranthene anions) that are produced in a negative-ion chemical ionization source and then brought into contact, in the gas phase, with multiply-charged positive ions that have been generated by ESI and trapped in a cell. Because of the excellent results that we had obtained by ECD and EDD of glycans, we undertook a new project to explore the utility of ETD for analysis of glycans and glycoconjugates. Since we did not yet have an instrument equipped for ETD in the Resource, we began our studies by taking advantage of an offer from Bruker Daltonics, Inc., who had an appropriately equipped quadrupole ion trap instrument available at their headquarters in nearby Billerica, MA. Our initial experiments produced spectra that have rich patterns that include both glycosidic and cross-ring cleavages. They therefore bear strong similarities to the ECD spectra of glycans that we have already obtained, but also exhibit additional fragments, and thus the data is complementary. In April 2009, Bruker installed one of their new ion trap instruments (amaZon ETD) in our laboratory, so that we may continue these studies and also generate reference tandem mass spectra for glycans that we will make available for inclusion in public databases. The mass spectra obtained by CID and ETD, alone and in combination, of a set of milk glycans have been obtained, for the native compounds, and for the permethylated derivatives of the native, reduced and O-18 labelled analogs. These were exhaustively interpreted and the resulting manuscript was published in the Journal of the American Society for Mass Spectrometry. Further analyses have included N-linked glycans and glycopeptides. In addition, variation of the chemical reagent for ETD and the chemical ionization gas is being evaluated to optimize the approach for glycans.

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National Center for Research Resources (NCRR)
Biotechnology Resource Grants (P41)
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