Date of Award

Winter 12-12-2022

Embargo Period

12-12-2022

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Cell and Molecular Pharmacology and Experimental Therapeutics

College

College of Graduate Studies

First Advisor

Richard Drake

Second Advisor

Anand Mehta

Third Advisor

Peggi Angel

Fourth Advisor

Shikhar Mehrotra

Fifth Advisor

Carl Atkinson

Abstract

Biofluids are a great source of biomarkers because they reflect the immune and metabolic status of cells throughout the body and can be collected non-invasively. Changes in the levels and compositions of N-glycans released from serum and plasma glycoproteins have been assessed in many diseases across many large clinical sample cohorts. Assays used for N-glycan profiling in these fluids currently require multiple lengthy processing steps, thus diminishing their potential for use as standard clinical diagnostic assays. In response to this need for rapid, simple, and high-throughput biofluid analysis, a novel slide-based biofluid profiling platform was developed for the detection of N-glycan alterations that can function as biomarkers of disease. This platform was initially validated for the analysis of serum and plasma N-glycan profiles but was also adapted for the evaluation of urine and prostatic fluid N-glycan profiles. Key to these workflows was the immobilization of the fluid glycoproteins to a slide that was washed of all contaminants, followed by the rapid and highly sensitive detection of enzymatically released N-glycans by mass spectrometry. The enzyme used to release the N-glycans can be changed to target and increase the sensitivity for specific N-glycan classes. To demonstrate the utility and feasibility of applying this platform, serum samples from 199 women with breast cancer and 99 women with a benign lesion in their breast were analyzed in order to identify differences in the N-glycan profiles. The overall N-glycan profiles of the two patient groups had no differences, but there were several individual N-glycans with significant differences in intensities between patients with benign lesions and ductal carcinoma in situ (DCIS). For women aged 50 - 74 with a body mass index of 18.5 - 24.9, a model including the intensities of two N-glycans, 1850.666 m/z and 2163.743 m/z, age, and BMI were able to clearly distinguish the breast cancer patients from the patients with benign lesions with an AUROC of 0.899 and an optimal cutoff with 82% sensitivity and 84% specificity. This study indicates that serum N-glycan profiling is a promising approach for providing clarity for breast cancer screening, especially within the subset of healthy weight women in the age group recommended for mammograms. Overall, the workflows described in this dissertation have displayed the sensitivity, adaptability, and throughput to be utilized as a biomarker discovery platform with significant clinical utility.

Rights

Copyright is held by the author. All rights reserved.

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