Multidimensional chromatography and mass spectrometry for differential glycoproteomics
Abstract
The goal of the study is to develop multidimensional chromatography and mass spectrometry methods for differential glycoproteomics. Glycopeptides from human transferrin and bovine fetuin were characterized by enzymatic digestion and mass spectrometry analysis. Two matrices were tested and compared in order to reduce the glycan fragmentation in MALDI-TOF MS analysis. Glycan structures were elucidated by glycosidase digestion or proposed by molecular masses of the glycans. The differences in sialylation across the proteome and the degree of sialylation within glycoproteins were examined using human serum samples. Sialylation was targeted through lectin affinity selection and quantification of differences was achieved with stable isotope coding in combination with reversed-phase chromatography (RPC) and mass spectrometry. The sialylation between a normal individual and pooled human serum varied less than two fold in all but 4–5 glycoproteins. The degree of sialylation among Con A selectable peptides was quite high. There can be substantial variation in sialylation between sites in the same protein. A comparative analysis of sialylated glycoforms with differentially branched complex type glycans was carried out by a simple and efficient approach based on the serial lectin affinity chromatography (SLAG) in combination with stable isotope labeling, RPC and mass spectrometry. The results showed that most sialylated glycopeptides identified carried more bianntennary glycans than tri- and tetraantennary glycans. The relative amount of biantennary glycan versus tri- and tetraantennary glycans might also vary at different glycosylation sites within the same glycoprotein. An alternative approach for identification of glycoproteins from human serum was developed based on a combination of lectin affinity chromatography and anion exchange/Cu-IMAC affinity selection. The approach can be used as an alternative method for isolation and identification of glycoproteins when lectins cannot effectively select glycopeptides.
Degree
Ph.D.
Advisors
Regnier, Purdue University.
Subject Area
Analytical chemistry
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