This application outlines methodology that may provide a detailed carbohydrate sequence, including monomer residue, linkage, and branching, at the femtomole level. The sequence strategy comprises three basic elements: (i) chemical modification of oligosaccharide residues to enhance sensitivity and induce greater molecular specificity; (ii) purification of modified structures by supercritical fluid chromatography (SFC); and, (iii) structural characterization of eluting components by on-line mass spectrometry (MS) using negative ion chemical ionization (NCI), collision induced dissociation (CID), and array detection (AD); i.e., SFC-NCI-CID- MS/MS-AD. Data are presented to support major aspects of this combined chemical, chromatographic and mass spectral approach, but additional effort must be undertaken to tie these components into a cohesive general strategy. This proposal outlines this effort with a focus on N-linked glycans by: (a) a study of alternative chemical methods for aldehyde trapping that could provide structural analogs with improved SFC chromatographic properties; (b) an introduction of more polar SFC mobile phases for the chromatography of unblocked, more complex, higher molecular weight glycans, peptides and glycopeptides; (c) developing comparable femtomole sensitivity (SFC-NCI-MS) with PFBAB-labeled, partially methylated and acetylated monosaccharides in support of monomer identification studies; and, (d) initiating high and low energy CID studies with the ZAB- T-AD and the proposed triple quadrupole instrument. Each of these areas are discussed with proposed methodology that would further enhance oligosaccharide sensitivity and extend structural detail.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM045781-02
Application #
3305212
Study Section
Metallobiochemistry Study Section (BMT)
Project Start
1991-05-01
Project End
1994-04-30
Budget Start
1992-05-01
Budget End
1993-04-30
Support Year
2
Fiscal Year
1992
Total Cost
Indirect Cost
Name
Harvard University
Department
Type
Schools of Public Health
DUNS #
082359691
City
Boston
State
MA
Country
United States
Zip Code
02115
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