Date of this Version
Diabetes is a metabolic disease that can lead to the non-enzymatic glycation of serum proteins such as human serum albumin (HSA). Previous studies have indicated that glycation can affect the structure and function of these proteins. This dissertation describes the development of tools and techniques based on high performance affinity chromatography (HPAC) and multidimensional mass spectrometry to analyze the effects of glycation on the function and structure of HSA.
A major portion of this research involved the utilization of HPAC to examine the effect of glycation on the binding of three second-generation sulfonylurea drugs and one third-generation sulfonylurea drug. These studies were conducted with HSA containing various levels of glycation. Frontal analysis and zonal elution competition studies were used to profile the binding properties of the drugs at the major and minor binding sites on samples of normal HSA and glycated HSA. Various trends in the binding affinity were observed for these drugs at the levels of glycation that were examined.
A second portion of this research involved the development of an on-line immunoextraction format in HPAC for examination of drug-protein interactions with normal and glycated HSA. This study utilized a polyclonal anti-HSA antibody HPAC column to extract and bind normal HSA or glycated HSA. The adsorbed HSA or glycated HSA columns were then tested and used in a number of chromatographic formats to examine drug-protein interactions.
Finally, a third portion of this research involved the use of multidimensional mass spectrometry to qualitatively profile the structure of HSA through sequence analysis. This work obtained sequence analysis results that were comparable to those found in a previous method involving matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. In addition, collision-induced dissociation was used to confirm the identity of several peptide sequences that could be used as internal calibrants for future work involving glycated HSA.
Advisor: David S. Hage