Application of Mass Spectrometry in Biology and Physiology
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Application of Mass Spectrometry in Biology and Physiology A dissertation submitted to the Graduate School of the University of Cincinnati in Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY (Ph.D.) In the Department of Chemistry of McMicken College of Arts and Sciences by Jiawei Gong Bachelor of Science (B.S.), Chemistry Xiamen University, 2012 Dissertation Advisor: Joseph A. Caruso, Ph.D Abstract Mass spectrometry, as an analytical technique that sorts ions based on their mass to charge ratio, is playing significant roles in analysis not only in chemistry field, but also in other areas such as biology and physiology. In this dissertation, the application of mass spectrometry, including both atomic and molecular mass spectrometry, was investigated in those two areas mentioned above. Inductively coupled plasma mass spectrometry (ICPMS), a typical atomic analysis technique, is a powerful tool for elemental detection and speciation. Instrumental advances, such as the dynamic reaction cell and triple quad alignment, gave rise to monitoring sulfur and phosphorus that suffered a lot from polyatomic interferences and ionization issue in previous study. Additionally, ICPMS is capable of element specific detection, where the intensity of each element is directly proportional to the element present in the samples, allowing for element quantification via peak area integration. All these capabilities mentioned above opened a new window for the detection and quantification of DNA protein crosslinks (DPCs) as there is always sulfur in proteins and phosphorous in DNA. In this dissertation, an approach for purification and quantitative analysis of DPCs was established by increasing the sensitivity of sulfur and phosphorus signal via ICPMS. Further structural determination was completed by electron spray ion trap mass spectrometry (ESI-ITMS), a typical molecular analysis technique for DNA or proteins. This new approach achieved the quantitative analysis of DPCs with low i detection limit and high sensitivity and enabled the identification of the binding sites on the protein involved in the crosslinking reaction. There are a variety of endogenous compounds affecting the cellular functions. Purification and characterization of these compounds laid the foundation for the further research on physiology. Endogenous cardiotonic steroids (CTS) were considered important for human health as inhibitors of the Na+/K+-ATPase. In this dissertation, an approach was established to purify the CTS from pig skeletal muscles including batch affinity extraction (BAE), gel filtration separation and reverse phase chromatography. NADH coupled method was used to checked the activity of the each fraction after sequences of purification. The molecular weight of the active compounds was determined by ESI-ITMS. This new approach with the combination of serious separation methods saved time and labor even though 2D nuclear magnetic resonance (NMR) analysis is still needed for the structural determination. ii iii Acknowledgement I would like to acknowledge many people for helping me during my Ph.D. studies. First of all, I would like to give special thanks to my research advisor, Dr. Joseph Caruso for his tutorage and guidance. As a successful scientist and chemist himself, his is always encouraging me to think and work form the scientific perspective. In addition, his kindness and optimistic enthusiasm comfort me a lot, especially in the first year I came to Cincinnati. Unfortunately, he passed away at the end of 2015. However, his smile will stay in my memory forever. Hope him rest in peace in the other world. I would like to express my deep appreciation to my committee members: Dr. Landero, who trained me from beginning and helped me grow to an analytical chemist; Dr. Merino, who always inspire me by new ideas and explained biological issues in details; Dr. Zhang, who treats me as a friend; Dr. Guan, who is always helpful and encourage me a lot. Also, Dr. Heiny gave me a lot of support during the collaboration. I would like to thank my colleagues in my research group, Cory Stiner, Skyler Smith, Keaton Nahan, Ryan Saadawi, Megan Schmale, Traci Hanley, Amberlie Clutterbuck, Anna Donnell, Nicole Hanks, for all the help they provided to me. iv I am also grateful to the department of chemistry, University of Cincinnati, for the financial support in the form of teaching assistantship and to Agilent technologies for the instrument support. v Table of Contents Chapter 1 Introduction 1.1 Introduction to research 2 1.2 Application of mass spectrometry in a biochemistry model study: DNA protein crosslinks characterization using model molecules under UV radiation 3 1.2.1 Introduction 3 1.2.1.1 Formation of DPCs 4 1.2.1.1.1 Formaldehyde-induced DPCs 4 1.2.1.1.2 Cisplatin-induced DPCs 5 1.2.1.1.3 Metal-induced DPCs 6 1.2.1.1.4 Reactive oxygen species (ROS) -induced DPCs 7 1.2.1.2 Biological consequences of DPCs 8 1.2.1.3 Enzymatic repair of DPCs 8 1.2.1.4 Detection of DPCs 10 1.2.2. Research Overview 12 1.2.2.1 Overview of Chapter 2 12 1.2.2.2 Overview of Chapter 3 13 1.3 Application of mass spectrometry in physiology study: cardiotonic steroids project 14 1.3.1 Introduction 14 1.3.3.1 Na+/K+-ATPase 14 1.3.3.2 Compounds displaying ouabain-like activity: Cardiotonic Steroids (CTS) 15 1.3.3.3 Purification of CTS 18 1.3.2 Overview of Chapter 4 18 vi Chapter 2 Simultaneously Detection and Quantification of synthesized DNA Protein Crosslinks by Triple Quad ICPMS (ICPQQQ) 2.1 Introduction 27 2.2 Experimental 31 2.2.1 Materials 31 2.2.2 Methods 31 2.2.2.1 DNA-Ribonuclease A Oxidative Cross-Linking 31 2.2.2.2 Optimization of the reaction in the octopole cell and other parameters 32 2.2.2.3 Quantification of sulfur and phosphor by using cap-RPHPLC-QQQ-ICPMS 33 2.2.2.4 Purification of the DPCs (DNA-Ribonuclease A cross-links) 33 2.2.2.5 Trypsin proteolysis of the DNA-Ribonuclease A cross-links 34 2.2.2.6 Pepetide identification after trypsin proteolysis by cap-RPLC-QQQ-ICPMS 35 2.3 Results and Discussion 35 2.3.1 Optimization of the instrument parameters 35 2.3.2 Quantification of S and P standards 37 2.3.3 Basic quantification and evaluation of laboratory synthesized DPCs 38 2.3.4 Cap HPLC-ICPMS detection of DPCs after trypsin proteolysis 39 2.4 Conclusions 40 Chapter 3 Structurally Characterization of DNA Protein Crosslinks (DPCs) by nanoLC-ESI-MS 3.1 Introduction 49 3.2 Experimental 52 vii 3.2.1 Materials 52 3.2.2 Methods 53 3.2.2.1 2’-Deoxyguanosine-Lys derivative Oxidative Cross-Linking 53 3.2.2.2 Purification of the dG-AcLysOMe adduct by RP-HPLC 53 3.2.2.3 Structure determination of the dG-AcLysOMe adduct by ESI-ITMS 54 3.2.2.4 Thermal stability study of the cross-linking covalent bond 55 3.2.2.5 DNA-Ribonuclease A Oxidative Cross-Linking 55 3.2.2.6 Purification of the synthesized DPCs 56 3.2.2.7 Thermal Hydrolysis of DNA-Ribonuclease A cross-links and purification of dG-Ribonuclease 56 3.2.2.8 Trypsin proteolysis of the dG-Ribonuclease A 56 3.2.2.9 Peptide separation and identification by HPLC-Chip-ESI-ITMS 57 3.2.2.10 MASCOT protein data base search 58 3.2.2.11 Calculations 58 3.3 Results and Discussion 59 3.3.1 A model DPC: the dG-AcLysOMe adduct 59 3.3.2 Thermolysis of the dG-AcLysOMe adduct 60 3.3.3 Purification of laboratory synthesized DPCs by SEC 61 3.3.4 Expansion to identification of whole protein DPCs 61 3.3.5 Identification of the dG-containing peptide-by-peptide sequence matching 62 3.3.6 Cross-linking prediction 64 3.4 Conclusion 64 Chapter 4 Purification and Identification of Novel Endogenous Cardiotonic Steroids (CTS) from pig skeleton muscle 4.1 Introduction 79 viii 4.2 Experimental 83 4.2.1 Materials 83 4.2.2 Methods 84 4.2.2.1 Homogenization of pig skeletal muscle 84 4.2.2.2 Batch affinity extraction 84 4.2.2.3 Post affinity gel filtration chromotography 85 4.2.2.4 Reverse Phase separation of OLC 86 4.2.2.5 Activity determination of OLC by NADH coupled essay 87 4.2.2.6 Mass spectrum analysis of active OLC fraciton 88 4.3 Results and Discussion 89 4.3.1 Post affinity gel filtration 89 4.3.2 Reverse Phase separation of OLC 90 4.3.3 Activity determination of OLC by NADH coupled essay 90 4.3.4 Mass spectrum analysis of active OLC fractions 91 4.4 Conclusion 92 Chapter 5 Future Directions 5.1 Future directions for DPCs project 110 5.2 Future directions of OLC project 112 ix List of Figures Chapter 1: Figure 1.1 Preferably targeted sited within DNA in the crosslinking reaction Figure 1.2 Mechanism of formaldehyde-induced DPCs Figure 1.3 Mechanism of cisplatin-induced DPCs Figure 1.4 Chelation mechanism of Cr-induced DPCs Figure 1.5 Structure of a) Marinobufagenin; b) ouabain; c) digoxin Chapter 2: Figure 2.1 Reaction Cell optimization for 47PO+ AND 48SO+ Figure 2.2 CapHPLC-QQQ-ICPMS chromatograms of organic standards Figure 2.3 SEC-QQQ-ICPMS chromatograms of reactants and solution after crosslinking reaction Figure 2.4 CapHPLC-QQQ-ICPMS Chromatograms from tryptic digested samples Chapter 3: Figure 3.1 Reverse Phase chromatograms of AcLysOMe, 2-dG reations Figure 3.2 MS & MS2 of the dG-AcLysOMe crosslinking compound Figure 3.3 MS of the dG-AcLysOMe crosslinking compound after heating at 170 oC for 1 hour Figure 3.4 Crosslinking reaction between 2-deoxguanosine (dG) and N-acetyl-Lysine-Methyl ester (AcLysOMe) Figure 3.5 Isoabsorbance plot after thermal hydrolysis of DPC x Figure 3.6 Mass spectrum for Identification of the dG-containing peptide by peptide sequence matching Figure 3.7 3D Structure of the Ribonuclease A Chapter 4: Figure 4.1