Advisor(s)

Barry L. Karger

Contributor(s)

Zhaohui S. Zhou

Date of Award

2010

Date Accepted

12-2010

Degree Grantor

Northeastern University

Degree Level

Ph.D.

Degree Name

Doctor of Philosophy

Department or Academic Unit

College of Arts and Sciences. Department of Chemistry and Chemical Biology.

Keywords

chemistry, lung cancer, biomarker, glycopeptides, LC-MS, monoclonal antibody

Subject Categories

Biochemical markers, Lungs - Cancer, Mass spectrometry, Liquid chromatography

Disciplines

Biochemistry | Cancer Biology | Medicinal-Pharmaceutical Chemistry

Abstract

Current proteomics strategies for biomarker efforts have two limitations: the neglect of protein glycosylation in discovery/qualification and the lack of antibodies for verification/validation. The main goal of this thesis is to address these two problems. For the lack of high quality antibodies, monoclonal antibody (mAb) proteomics - high throughput mAb generation and disease -specific mAb screening - was applied to lung cancer biomarker discovery. mAb proteomics is demonstrated to generate high quality mAbs in a high throughput manner, an important characteristic for biomarker verification/validation. For glycosylation characterization, mass spectrometry (MS)-based methods for sensitive, site-specific discovery and quantitation were developed and applied to characterize the glycosylation changes of the lung cancer biomarker candidate haptoglobin. These developed techniques for glycosylation characterization can be also applied to other glycoproteins and therefore will contribute to the emerging glyco biomarker field.

In Chapter 2, together with Biosystem International (BSI), we generated an mAb library using mAb proteomics against lung cancer patient plasma protein mixtures. Cancer specific mAbs were identified by high throughput screening, and three mAbs were produced in large quantity for antigen (Ag) identification purposes. An MS-based strategy combined with immunoassay was developed for Ag identification. One of the antigens was found to be plasma glycoprotein haptoglobin (Hpt). The anti-Hpt Ab-Ag interaction was characterized using various techniques, and the mAb was found to have higher affinity for native plasma Hpt than the corresponding reduced form. Furthermore, the glycan on the Hpt was found to be essential for the Ab-Ag interaction. The characterization of mAbs in this work revealed that the global mAb proteomics process can generate high-quality lung cancer specific mAbs capable of recognizing proteins in their native state.

In Chapter 3, a robust, ultrasensitive LC-MS platform, combining the strength of 10 µm i.d. porous layer open tubular (PLOT) LC column with linear ion trap-collision induced dissociation / electron transfer dissociation - mass spectrometry (LTQ-CID/ETD-MS), was successfully developed for site-specific protein glycosylation characterization. Using Hpt as a proof-of-concept model glycoprotein, glycopeptide identification, glycosylation site elucidation, glycan quantitation and site occupancy determination, were demonstrated to be completed within 10 LC-MS runs at a total sample consumption of 100 fmol (~ 13 ng Hpt). The developed platform was shown to have sufficient sensitivity to allow characterization of site-specific protein glycosylation from trace amounts of glycosylated proteins.

In Chapter 4, a glycopeptide selected reaction monitoring (SRM) method was developed to sensitively and reproducibly quantify protein glycosylation isoforms in a site-specific manner. A feasibility study was first performed to ascertain the potential of glycopeptide SRM analysis using Chip LC-quadrupole time-of-flight mass spectrometer (Chip LC-qToF) and Chip LC- triple quadrupole mass spectrometer (Chip LC-QQQ). Glycopeptide fragmentation pattern was systematically studied, and glycan oxonium ions were determined to be preferred SRM product ions. The glycopeptide SRM method featured high sensitivity, high reproducibility and large dynamic range. Site-specific glycosylation differences of Hpt, affinity purified from individual non-small cell lung cancer, healthy controls and inflammation controls (rheumatoid arthritis) plasma samples, were revealed using the glycopeptide SRM method. Further verification/validation of these discovered site-specific Hpt glycosylation isoforms may increase the specificity of Hpt as a lung cancer biomarker.

Document Type

Dissertation

Rights Holder

Dongdong Wang

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