Mass spectrometric determination of glycation sites in hemoglobins

CHED 342

Billy M Clifford-Nunn, bcliffor@umflint.edu, Department of Chemistry and Biochemistry, University of Michigan - Flint, 556 Murchie Science Building, 303 East Kearsley Street, Flint, MI 48502 and Sonja Hess, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, 8 Center Drive, Building 8, Room B2A19, Bethesda, MD 20892.
High extracellular levels of glucose lead to glycation of hemoglobins in diabetes patients therefore a long-term diagnostic tool is the analysis of glycated hemoglobins. Here we describe the mass spectrometric characterization of hemoglobin subunits and identification of glycation sites. Fetal hemoglobin standards, sickle cell hemoglobin standards, and glycated hemoglobin variants from diabetic patients were isolated. The alpha, beta, sickle, gamma, and glycosylated chains were identified using reverse phase liquid chromatography (LC), strong cation exchange (SCX) and tandem mass spectrometry (MS/MS) technologies. The main peaks on the chromatograms were assigned to separate subunits according to their molecular weights. Each fraction was digested using trypsin and the peptides obtained were sequenced using LC-MS/MS. To verify known peptides, database searches were performed. From these searches the appropriate peaks were labeled with the appropriate subunit. Using this method, known glycation sites were verified including glycation sites on the N-termini of alpha and beta chains.
 

Undergraduate Research Poster Session: Analytical Chemistry
11:00 AM-1:00 PM, Monday, March 26, 2007 Hyatt Regency Chicago -- Riverside Center, Poster

Division of Chemical Education

The 233rd ACS National Meeting, Chicago, IL, March 25-29, 2007