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Development and Application of Liquid Chromatography and Mass Spectrometry Platforms to Cancer Samples Advances in Glycoproteomics and Glyco-Biomarker Discovery Studies: Development and Application of Liquid Chromatography and Mass Spectrometry Platforms to Cancer Samples by Francisca Owusu Gbormittah B.S. in Chemistry, Kwame Nkrumah University of Science and Technology M.S. in Chemistry, Indiana University of Pennsylvania A dissertation submitted to The Faculty of the college of Science of Northeastern University in partial fulfillment of the requirements for the degree of Doctor of Philosophy August 1, 2014 Dissertation directed by William S. Hancock Professor of Chemistry and Chemical Biology UMI Number: 3633335 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. UMI 3633335 Published by ProQuest LLC (2014). Copyright in the Dissertation held by the Author. Microform Edition © ProQuest LLC. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, MI 48106 - 1346 DEDICATION To my family ii ABSTRACT The development of analytical technologies to investigate the glycoproteome of clinical relevant samples has improved over the last 10 years. These new developments aim to improve the identification and quantification of disease-specific glyco-biomarkers, which are present at low amounts in biological matrices. Glyco-biomakers have the potential to significantly contribute to cancer discovery studies in specific areas such as; early diagnosis, prognosis, monitor cancer recurrence and improve the low survival rate of cancer. In this thesis, we focused on the development and application of novel liquid affinity chromatography fractionation platforms integrated with nano-LC-MS/MS to characterize and quantify the glycoproteome as well as selected glyco-biomarker candidates of cancer samples. In chapter 1, brief background information covering glycoproteomics and glyco- biomarker discovery studies is presented. Specifically, protein glycosylation process and how the field of ‘omics’, which includes glycoproteomics, have revolutionized clinical glyco-biomarkers discovery are discussed. Further, various disease models, current sample fractionation strategies and analytical methodologies involved in glyco-biomarker development pipeline and their significance as well as their short falls are described. Reviewing biomarker validation and current bio-infomatics tools utilized in glycoproteomics discovery studies concludes chapter 1. Chapter 2 details the development of a novel multi-dimensional affinity liquid chromatography fractionation approach that combines the depletion of the top 12 abundant proteins and multi-lectin fractionation of the human plasma. Evaluating and validating the reproducibility, specificity and overall recoveries of the platform demonstrated the suitability of the developed method in glyco-biomarker discovery studies of clinical samples. After establishing this robust platform, it was applied in chapter 3 to comprehensively study the global glycoproteome profile of clear cell renal cell carcinoma plasma (ccRCC) samples to identify and iii characterize potential biomarkers for early detection of the disease. During this study, protein abundance alterations as well as glycan shifts were investigated to understand the sub-proteome of ccRCC. Chapter 4 focuses on the structural characterization of a glycoprotein (clusterin) that was identified during the ccRCC biomarker discovery studies. Clusterin has been implicated in ccRCC cancer progression however; its structure and biological function(s) are not yet well defined. Therefore, to have more structural insights into clusterin, the protein was immuno- affinity purified from ccRCC plasma followed by tandem mass spectrometry to profile glycoforms, N-glycosylation sites and quantify glycan amounts. We discovered that the levels of bi-antennary digalactosylated disialylated (A2G2S2) and core fucosylated bi-antennary digalactosylated disialylated (FA2G2S2) glycans differed significantly in the plasma of patients before and after curative nephrectomy of localized ccRCC. In chapter 5, a multi-lectin affinity chromatography platform previously developed in our laboratory was optimized and applied to investigate glycoproteins and non-glycoproteins present in pancreatic cyst fluid samples. This study was aimed at identifying potential candidate markers for early detection of malignant cyst (pancreatic cancer precursor). Our data showed the identification of proteins with significant differential expression in mucinous cysts (malignant cyst) compared to non-mucinous cysts (benign) of which one protein (periostin) associated with cancer progression was confirmed by immunoblotting assay. In the final chapter (chapter 6), we summarize and conclude our findings in this work and provide our perspective on the potential of glycoproteins in glyco-biomarker discovery studies. iv TABLE OF CONTENTS Page # DEDICATION ii ABSTRACT iii TABLE OF CONTENTS vi APPENDICES xii LIST OF FIGURES xiii LIST OF TABLES xv CHAPTER 1 1 INTRODUCTION: OVERVIEW OF GLYCOPROTEOMICS AND GLYCO-BIOMARKER DISCOVERY STUDIES 1.1 Protein glycosylation and glycoproteomics 2 1.2 Current status of glyco-biomarkers: advantages and limitations 3 1.3 Biological matrices 4 1.3.1 Blood plasma/serum 4 1.3.2 Tumor tissue 5 1.3.3 Proximal fluids 5 1.3.4 Tumor cell lines 6 1.4 Glycoproteomics sample fractionation strategies 6 1.4.1 Lectin affinity platforms and applications 7 1.5 Glycoproteomics characterization using Mass Spectrometry (MS) approaches 9 1.5.1 Proteolytic enzymes selection 10 v 1.5.2 Glycoproteins and glycopeptides enrichment strategies 10 1.5.3 MS spectrometry platform 11 1.6 Quantitative technologies in glycoproteomics 15 1.6.1 Stable isotope quantitation 15 1.6.2 Targeted-based quantitation 16 1.6.3 Label free quantitation 16 1.7 Glyco-biomarker validation strategies 17 1.8 Data Processing and Statistical Analysis of Glycoproteomics 18 1.9 References 20 CHAPTER 2 30 DEVELOPMENT OF AN IMPROVED FRACTIONATION OF THE HUMAN PLASMA PROTEOME BY A COMBINATION OF ABUNDANT PROTEINS DEPLETION AND MULTI-LECTIN AFFINITY CHROMATOGRAPHY 2.1 Abstract 31 2.2 Introduction 32 2.3 Materials and Methods 35 2.3.1 Materials 35 2.3.2 Samples for study 36 2.3.3 Experimental design 36 2.3.4 Preparation of 12P, M-LAC and reverse phase HPLC columns 37 2.3.5 High abundance protein depletion and multi-lectin affinity Fractionation 38 2.3.6 Protein concentration measurements and 1D-SDS PAGE analysis 39 2.3.7 In-solution protein trypsin digestion 40 2.3.8 Nano-LC-MS/MS analysis and peptide sequencing 41 vi 2.4 Results and discussion 43 2.4.1 12P immuno-affinity depletion 43 2.4.2 Specificity of 12P depletion column 46 2.4.3 12P-M-LAC fractionation platform 46 2.4.4 Recovery studies of 12P-M-LAC platform 47 2.4.5 Reproducibility studies of protein identification from the 12P-M-LAC platform fractions 47 2.4.6 Enrichment of low level glycoproteins by 12P-M-LAC platform 51 2.5 Conclusion 53 2.6 References 54 CHAPTER 3 57 COMPARATIVE STUDIES OF THE PROTEOME, GLYCOPROTEOME AND N-GLYCOME OF CLEAR CELL RENAL CELL CARCINOMA PLASMA BEFORE AND AFTER CURATIVE NEPHRECTOMY 3.1 Abstract 58 3.2 Introduction 59 53.3 Materials and Methods 61 3.3.1 Materials 61 3.3.2 Sample population 62 3.3.3 High Abundance Proteins Depletion and Glycoprotein Affinity Fractionation 63 3.3.4 N- Glycan Release and LC-ESI-MS Analysis 64 3.3.5 Gel nano-LC-MS/MS Proteomic and Glycoproteomic Analysis 65 3.3.6 Data processing and statistical analysis 66 3.4 Results and discussion 68 vii 3.4.1 The analytical strategy 68 3.4.2 The 12P-M-LAC analytical platform 70 3.4.3 Overview of proteomics and glycoproteomics data 71 3.4.4 Quantification and selection of differentially expressed proteins present in 12P depleted ccRCC plasma proteome 72 3.4.5 Identification and selection of proteins of interest showing differential M-LAC column binding 76 3.4.6 Characterization of N-glycan moieties released from depleted M-LAC fractions by porous graphitized carbon (PGC) LC-ESI-IT MS/MS 79 3.4.7 N-glycan structures alteration analysis 80 3.4.8 Validation of differentially expressed N-glycans by extracted ion Chromatograms 84 3.5 Conclusion 86 3.6 References 87 CHAPTER 4 92 TANDEM MASS SPECTROMETRY CHARACTERIZATION OF CLUSTERIN GLYCOPEPTIDE VARIANTS IN THE PLASMA OF CLEAR CELL RENAL CELL CARCINOMA 4.1 Abstract 93 4.2 Introduction 94 4.3 Materials and Methods 96 4.3.1 Materials 96 4.3.2 Clear cell renal cell carcinoma (ccRCC) plasma sample collection and preparation 97 viii 4.3.3 Clusterin immuno-affinity HPLC purification 97 4.3.4 Lectin blot assay of purified Clusterin 98 4.3.5 One dimensional SDS PAGE and enzymatic digestion 98 4.3.6 C18 reversed phase nano-LC-MS/MS Analysis 99 4.3.7 Data and statistical Analysis 100 4.4 Results and discussion 101 4.4.1 Development of the Analytical Approach 101 4.4.2 Glycan occupancy analysis 106 4.4.3 Characterization of site-specific oligosaccharide heterogeneity 109 4.4.4 Glycan structures for the selected glycopeptide residue 372-385, N-374 112 4.4.5 Quantitation of targeted glycoforms in clinical samples 115 4.4.6 Lectin blot assay 119 4.5 Conclusion 120 4.6 References 122 CHAPTER 5 128 CHARACTERIZATION OF GLYCOPROTEINS IN PANCREATIC CYST FLUID USING A HIGH PERFORMANCE MULTIPLE
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