A targeted glycoproteomics for breast cancer associated glycoproteins
Abstract
The major objective of the work presented in this thesis was to develop a global targeted glycoproteomics method using lectin and glycan targeting antibodies for the identification of breast cancer associated glycoproteins in human plasma. A secondary focus was to recognize the concentration changes of glycosylation in a disease state. An analytical tool for glycoproteomics was developed for the selection of glycoproteins in normal and human breast cancer patient plasma. In order to identify more glycoproteins, Buttom-Up approaches (BUA) and Top-Down approaches (TDA) were examined. More proteins were identified by the Top-Down approach than Buttom-Up approach. Based on these results, TDA was applied in the targeted glycoproteomics studies. Lectin affinity chromatography using concanavalin A (Con A), Helix pomatia agglutinin (HPA), Lycopersicon esculentum lectin (LEL), Aleuria aurantia lectin (AAL) and Lens culinaris agglutinin (LCA) was used to investigate the utility of narrow selectivity lectins in the characterization of plasma glycoproteome diversity and to recognize cancer associated aberrations in glycosylation. Following affinity chromatographic selection, proteins were tryptically digested, the peptide fragments separated by reversed phase chromatography (RPC), and fractions from RPC identified by tandem mass spectrometry. The diversity of glycosylation found with narrow selectivity lectins was generally 2/3 that of Con A and not related to protein abundance. Small groups of proteins were found with each of the affinity columns, HPA, LEL, AAL, and LCA, that changed 3-fold or more in concentration between normal and breast cancer patient plasma. Although the number of cancer patients examined was too small to validate cancer marker candidates, they are clearly worth examining in a larger, more diverse patient population. Immunoaffinity chromatography (IAC) was also used to isolate and identify potential cancer biomarker glycoproteins by targeting disease-associated glycans. Glycoproteins were selected from plasma of disease-free and breast cancer patients with an anti-sialylated Lewis x (sLex) IAC column. After extensive washing of the IAC column to remove abundant proteins, the selected proteins were eluted with an acidic mobile phase and identified in two ways. The protocol used in route A involved the steps of tryptic digestion, reversed-phase chromatographic fractionation of the digest, and identification of peptides in collected RPC fractions by MALDI-MS/MS. Route B differed in that IAC selected proteins were further fractionated by reversed phase chromatography before proteolysis of individual chromatographic fractions and identification by MALDI MS/MS. Route A was the more efficacious of the two protocols in total number of proteins identified. Ig gamma-1 chain C region, Ig mu chain C region, vitronectin, histidine-rich glycoprotein, inter-alpha-trypsin inhibitor heavy chain H4, and proteoglycan-4 changed three fold or more in association with breast cancer. The potential of these candidates as cancer markers remains to be validated in much larger, more diverse populations of breast cancer patients.
Degree
Ph.D.
Advisors
Regnier, Purdue University.
Subject Area
Analytical chemistry|Biochemistry|Organic chemistry
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