The development of serial lectin affinity techniques for the study of O-glycosylation in healthy and disease states

The major goal of the work presented in this thesis was the development of serial lectin affinity chromatography (SLAC) methods for the selection and analysis of O-glycoproteins and peptides. This study began by combining the lectins Concanavalin A (Con A) and Jacalin in series to target the most common O-glycan core (Gal-(β, 1-3)-GalNAc). Reverse phase chromatography and mass spectrometry were combined with this SLAC method for the selection and identification of O-glycopeptides from fetuin. After successful validation, this multidimensional chromatography/mass spectrometry approach was applied to human serum. After the completion of the above research it was discovered that several proteins with large amount of N- and O-glycosylation were not being selected. To combat this problem a SLAC method was developed which used Con A to select N-glycoproteins and Jacalin to select the resulting O-glycopeptides. The next phase of this research was the selection of additional carbohydrates. O-glycans have eight known core structures which can be decorated with various monosaccharides including GlcNAc and sialic acid. By using WGA (specific for GlcNAc) and SNA (specific for sialic acid) to select O-glycopeptides with these carbohydrates a better picture of O-glycosylation in human serum was developed. The final phase in this research was the application of the original SLAC method to arthritis in humans in order to determine with O-glycosylation is associated with this disease. By comparing normal pooled serum with three different forms of arthritis it was found that several proteins had significant concentration changes