Characterization of affinity ligands by MALDI-TOF MS and the preparation of affinity restricted access media

The first topic considered in this dissertation was an investigation of heterogeneous protein ligands using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Human serum albumin (HSA) was used as a model in these studies. A means to obtain high sequence coverage in protein studies by MALDI-TOF MS was developed by optimizing matrix preparation and using multiple enzyme digests with peptide fractionation. The optimized method could be used to examine unique peptides from nearly all of the structure of HSA and to identify specific modifications to this protein. This MALDI-TOF MS method was applied in quantitative studies of heterogeneous immobilization sites on HSA that had been coupled to silica through the Schiff base method. The immobilized HSA and soluble HSA were digested separately in normal water or 18O-enriched water and then mixed together for analysis by MALDI-TOF MS. Several peptides were found to have significantly higher 18O/16O ratios than other peptides in the same digests, implying their involvement in immobilization. Analysis of these results led to identification of the N-terminus and several lysines as likely immobilization sites for HSA. MALDI-TOF MS was also applied in studying the inherent heterogeneity in minimally glycated HSA. By comparing the peptide maps of normal HSA and glycated HSA, twelve lysines and three arginines were found to be likely modification sites in minimally glycated HSA. The modifications on these sites were also identified by employing their observed mass shifts. The second general topic of this dissertation focused on the development of affinity restricted access media using antibodies as immobilized ligands. Rabbit IgG and anti-fluorescein antibodies were used as model ligands in this work. These antibodies were first immobilized onto silica supports, with ligands on the outer surface being cleaved by an enzyme while antibodies in the support pores were left intact. Items evaluated in the development of such media included the immobilization method, pore size of support, the type of enzyme used support treatment, the levels of this enzyme level and the digestion time. The optimized conditions were used to prepare the anti-fluorescein antibodies restricted access media columns for ultrafast immunoextraction. Injections of fluorescein and fluorescein-labeled bovine serum albumin on this column indicated that the resulting stationary phase had high selectivity for small versus large molecules containing the same binding regions for antibodies