In-depth proteomics and glycoproteomics by mass spectrometry and affinity-based platforms for selectively capturing proteins and glycoproteins from breast cancer and disease free human serum

Abstract

Scope and Method of Study:

The major purpose of the work presented in this dissertation is to contribute to the introduction and evaluation of new ways and strategies for reducing the complexity of human serum and in turn contribute to facilitating the identification of candidate biomarkers with the assistance of advanced mass spectrometry techniques. In one approach, immobilized metal affinity chromatography (IMAC) and protein equalizer techniques were combined to reduce the complexity of the human serum. In another approach, lectin affinity chromatography involving tandem lectin columns with narrow and broad specificity lectins were evaluated in the selective enrichment of the glycoproteins present in human serum.

Findings and Conclusions:

The investigation described in this dissertation has significantly contributed to the in-depth proteomics analysis of human serum. The equalization/IMAC strategy allowed the identification of 82 non-redundant proteins, which was facilitated by the IMAC postfractionation process after equalization. The tandem-lectin affinity based platforms developed in this investigation selectively captured glycoproteins from breast cancer serum and disease-free serum and many proteins that were differentially expressed in the cancer serum were identified. In a platform where a combination of broad and narrow specificity lectins were evaluated in tandem series, 165 non-redundant proteins were identified. The platforms developed in this investigation are expected to be of general use and to facilitate the identification of additional candidate biomarkers for various diseases in the future.