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Infrared Laser Ablation for Biomolecule Sampling Kelin Wang Louisiana State University and Agricultural and Mechanical College, [email protected]

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Infrared Laser Ablation for Biomolecule Sampling Kelin Wang Louisiana State University and Agricultural and Mechanical College, Kelin.Wang641@Gmail.Com Louisiana State University LSU Digital Commons LSU Doctoral Dissertations Graduate School 3-18-2019 Infrared Laser Ablation for Biomolecule Sampling Kelin Wang Louisiana State University and Agricultural and Mechanical College, [email protected] Follow this and additional works at: https://digitalcommons.lsu.edu/gradschool_dissertations Part of the Analytical Chemistry Commons Recommended Citation Wang, Kelin, "Infrared Laser Ablation for Biomolecule Sampling" (2019). LSU Doctoral Dissertations. 4876. https://digitalcommons.lsu.edu/gradschool_dissertations/4876 This Dissertation is brought to you for free and open access by the Graduate School at LSU Digital Commons. It has been accepted for inclusion in LSU Doctoral Dissertations by an authorized graduate school editor of LSU Digital Commons. For more information, please [email protected]. INFRARED LASER ABLATION FOR BIOMOLECULE SAMPLING A Dissertation Submitted to the Graduate Faculty of the Louisiana State University and Agricultural and Mechanical College in partial fulfillment of the requirements for the degree of Doctor of Philosophy in The Department of Chemistry by Kelin Wang B.S., Liaoning University of Petroleum and Chemical Technology, 2009 M.S., Western Kentucky University, 2012 May 2019 To my loving parents ii ACKNOWLEDGEMENTS The most important person I must thank first is my major advisor, Dr. Kermit K. Murray, my committee chair, major advisor and doctoral mentor, for his wisdom, caring, and support during this endeavor. The constant encouragement he awarded me to build up confidence in myself and independent thinking skills on research problems. His sharp mind usually helps me through obstacles and hard time. It is impossible for me to finish this dissertation without his unwavering guidance and persistent help. I greatly appreciate the remaining members of my committee, Dr. Megan A. Macnaughtan and Dr. John A. Pojman for the time and valuable advice they provided generously and also for the good questions which incented me to widen my research from various perspective. Thanks to Dr. Fabrizio Donnarumma for valuable insights and help on biomolecular analysis, without which this work would be lacking and for which I am deeply thankful. Thanks to Dr. Scott W. Herke for the countless time assisting of genomic analysis. I would also like to express my gratitude to all the faculty and staff in LSU chemistry department, especially to Ms. Connie David and Dr. Jeonghoon Lee from LSU mass spectrometry facility, as well as Dr. Clayton Loehn from LSU shared instrument facility. Acknowledgements are also given to my colleagues, Fan Cao, Bijay Banstola, Chao Dong, Remi Lawal, Deenamulla Kankanamalage Achala Priyadarashani and Jamira Stephenson. Thanks for their assistance and help in my research work. iii Special thanks to Dr. Boliang Zhang, for all the precious encouragement and help during hard times, as well as all the good memories and happiness during these five years. This dissertation is dedicated to my parents and grandparents in China for their unconditional love and unbelievable support. iv TABLE OF CONTENTS ACKNOWLEDGEMENTS ........................................................................................................... iii LIST OF TABLES ........................................................................................................................ vii LIST OF FIGURES ..................................................................................................................... viii LIST OF ABBREVIATIONS AND SYMBOLS .......................................................................... xi ABSTRACT ................................................................................................................................. xiv CHAPTER 1 INTRODUCTION ................................................................................................. 1 1.1. LOCALIZED SAMPLING METHODS .................................................................. 3 1.2. BIOMOLECULE ANALYSIS .............................................................................. 11 1.3. OBJECTIVE ........................................................................................................... 21 CHAPTER 2 INSTRUMENTATION ....................................................................................... 22 2.1. INFRARED OPTICAL PARAMETRIC OSCILLATOR LASER ........................ 23 2.2. IR LASER ABLATION AND TRANSFER .......................................................... 24 2.3. ABLATION CONTROL SOFTWARE ................................................................. 26 2.4. LASER ABLATION WORKFLOW ..................................................................... 27 2.5 MALDI TOF/TOF MS ............................................................................................ 28 2.6 UPLC-HDMSE ........................................................................................................ 28 2.7 SANGER SEQUENCING ...................................................................................... 28 2.8 BIOANALYZER .................................................................................................... 29 2.9 PCR ......................................................................................................................... 30 2.10 QUBIT ................................................................................................................... 31 2.11 FLUORESCENCE READER ............................................................................... 31 2.12 TISSUE SAMPLES PREPARATION .................................................................. 31 2.13 CHEMICAL AND MATERIALS ........................................................................ 32 CHAPTER 3 MALDI DIRECTED LASER ABLATION TISSUE MICROSAMPLING WITH DIA MASS SPECTROMETRY ................................................................................................... 35 3.1. INTRODUCTION .................................................................................................. 35 3.2. EXPERIMENTAL ................................................................................................. 37 3.3. RESULTS AND DISCUSSION ............................................................................ 39 v 3.4. SUMMARY ........................................................................................................... 48 CHAPTER 4 INFRARED LASER ABLATION AND CAPTURE OF ENZYMES WITH CONSERVED ACTIVITY ........................................................................................................... 50 4.1. INTRODUCTION .................................................................................................. 50 4.2. EXPERIMENTAL ................................................................................................. 53 4.3. RESULTS AND DISCUSSION ............................................................................ 57 4.4. SUMMARY ........................................................................................................... 63 CHAPTER 5 INFRARED LASER ABLATION SAMPLE TRANSFER OF TISSUE DNA FOR GENOMIC ANALYSIS ...................................................................................................... 65 5.1. INTRODUCTION .................................................................................................. 66 5.2. EXPERIMENTAL ................................................................................................. 68 5.3. RESULTS AND DISCUSSION ............................................................................ 69 5.4. SUMMARY ........................................................................................................... 77 CHAPTER 6 RNA SAMPLING FROM TISSUE SECTIONS USING IR LASER ABLATION FOR QUANTITATIVE PCR ....................................................................................................... 79 6.1. INTRODUCTION .................................................................................................. 79 6.2. EXPERIMENTAL ................................................................................................. 82 6.3. RESULTS............................................................................................................... 84 6.4. SUMMARY ........................................................................................................... 91 CHAPTER 7: CONCLUSIONS AND FUTURE DIRECTIONS ................................................ 93 REFERENCES ............................................................................................................................. 96 APPENDIX. LETTERS OF PERMISSION ............................................................................... 123 VITA ........................................................................................................................................... 125 vi LIST OF TABLES Table 2.1 Sources for laboratory supplies..................................................................................... 34 Table 3.1 Summary of peptides and proteins identification in three tissue sections. ................... 45 Table 3.2 Summary of all MALDI peaks assigned based on LC-MS/MS analysis of the laser ablated extracts including MW of the identified proteins, MW difference (ΔM) between MALDI peaks and MW of identified proteins, Uniprot accession number and protein names ................. 46 Table 3.3 Full names of the proteins in Figure 3.5. .....................................................................
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