Analysis of modifications and behaviours of a hybrid beta-hexosaminidase and other biomolecules by mass spectrometry and liquid chromatography

Abstract

The study of post-translational modifications is a large focus in proteomics experiments as these modifications are often what drive signalling and trafficking of proteins within a cell or an organism. In the immune system, the glycosylation of immunoglobulins is a key point of the study of xenotransplantation. In lysosomes, the glycosylation or phosphorylation of both an enzyme and its substrate is highly important in degradation pathways, and will result in disease if interrupted. Immunoglobulin G and Tay-Sachs disease are respective examples of these instances, and both are discussed in detail in this thesis. Subtypes of porcine immunoglobulin G were studied by liquid chromatography and mass spectrometry in order to determine classification by glycoproteome analysis. Variations of these techniques, particularly electrospray-ionization and matrix assisted laser desorption/ionization mass spectrometry, were used to study the glycosylation and phosphorylation profiles of a hybrid beta-hexosaminidase enzyme which was designed to be an improved contender for enzyme replacement therapy for Tay-Sachs disease. The substrate of this enzyme, a ganglioside, was also analyzed using these techniques in order to determine detectability and relative quantification in mice brains. Liquid chromatography and mass spectrometry are the techniques most commonly utilized in proteomic and glycoproteomic experiments. Complex samples can be analyzed by nuanced techniques such as two-dimensional liquid chromatography, and this information can be used to create algorithms to aid in identification of post-translationally modified peptides. The Sequence-Specific Retention Calculator is an example of an algorithm which was created in part for this purpose, and it is explored in this thesis in two different experiments. Phosphorylated peptides are more difficult to detect in mass spectrometry, and only make up a small portion of peptides in a cell. Due to this, it is useful to utilize liquid chromatography to both simplify a proteomic sample and create another dimension in which identification can be performed. Development of these techniques includes the optimization of a mobile phase, which was explored using both formic and acetic acid.