Development of quantitative analytical methods for the reliable assessment of the roles of glycans in the development and progression of breast cancer

Though there are many post translational modifications made to proteins, glycosylation is one of the most common. Glycans play important biological roles and have key functions in processes like cell signaling and cell adhesion. The aberrant expression of glycans has been linked to diseases such as various types of cancer. Differences in glycan expression are key to discovering possible biomarkers for the diseases and the variations. While there are many ways in which glycans can be analyzed, liquid chromatography mass spectrometry is method that will be the main focus for the applications in this work. As ionization energy can fluctuate between sample runs and there is a need for high throughput methods of glycomic analysis, multiplexing is becoming a promising methodology for running glycomic analysis. There are many multiplexing strategies but many face complex issues like low reporter ion yield and labeling overlap while few stabilize the labile moieties in glycans. An 8-plex method using CH3I, CH2DI, CHD2I, CD3I, 13CH3I, 13CH2DI, 13CHD2I, 13CD3I that was previously published by our group avoids these problems and herein this work the method has been expanded to 16-plex through the use of 18O labeling. The 16-plex method was tested on standard glycoproteins through the use of the C18 liquid chromatography column the original 8-plex method was designed for. However, this work also tests to see if the 16-plex method can work on micro pillar array columns. As mentioned, aberrant glycosylation has been linked to cancer. This work specifically looks into the significant expression changes between the four major subtypes of breast cancer: luminal A, luminal B, HER2 positive, and triple negative. One hundred and twenty breast cancer samples were tested, thirty each coming from ten patients of each cancer subtype. The significant glycan expression differences between the subtypes were explored and possible candidates for glycan based biomarkers were found with which the cancer subtypes may be able to be differentiated. This work also outlines the future work needed for the projects discussed herein.

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