Mass spectrometry applications in glycoprotein analysis and identification of proteins by peptide mass fingerprinting methods
Woosley, Bryan David
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The increasing use of mass spectrometry (MS) has created a growing field of proteomic analysis that relies heavily on proper separation of complex mixtures and analysis so that sequencing of proteins and glycoproteins can be accomplished. Advances in computer sequencing algorithms and separation techniques have provided a wealth of data concerning the amino acid sequence of proteins. However, as more protein sequences have been identified, it is apparent that the amino acid sequence alone is not sufficient for predicting the function of the protein. Although the gene sequence coding for a specific amino acid is determined by the DNA of an organism, the protein that is made, and more specifically, the function of the protein that is made is determined by the modifications that take place after the protein sequence has been translated. Mass spectrometry has long been used to determine the amino acid sequence of a protein, but the post-translational modifications (PTMs) of a protein have been more challenging to analyze due to their complexity and the absence of a method that can be used to identify multiple modifications in one analysis. Of more than 200 types of PTMs, phosphorylation and glycosylation are two of the most important in terms of the effect that they have on the function of the protein. Because phosphorylation and glycosylation both have specific amino acid sites of modification, development of techniques to identify the presence or absence of such modification to those sites is critical in a full understanding of the protein structure and, consequently, its function. The focus of this work is centered around identifying sites of glycosylation on recombinant forms of Aspergillus niger endopolygalacturonase A and C. Although enzymatic treatment with PNGase F has widely been established as an effective method for determining sites of N-glycans on glycoproteins, there is no such enzyme to effectively remove O-glycans for specific analysis. This works uses a combination of beta-elimination followed by Michael addition (BEMAD) and electro-spray ionization (ESI) cone voltage switching to identify O-glycans. Presented in this work is the first application of using BEMAD to identify O-mannose sites on a protein, and a complete glycan analysis was performed as a result. This work also includes a study of the factors affecting the results of peptide mass fingerprinting (PMF) and the minimum coverage required to successfully identify a protein using these methods in addition to the affect of mass accuracy on these requirements.