ABSTRACT GANAWI, AMEL AHMED.A. Extraction and Direct Quantification of Salicylic Acid in Arabidopsis Using Matrix-Assisted Laser Desorption Ionization Mass Spectrometry. (Under the direction of Assistant professor Lin He). Systemic acquire resistant (SAR) is enhanced resistance created by the plant after infection, in which the plant becomes resistant to a second pathogen infection. This resistant can last for weeks preventing the plant from being infected. In addition the plant becomes resistant not only to the pathogen that causes the infection but also to a wide range of pathogens. Knowing how to activate this resistant in the plant is critical to increase crop production. Salicylic Acid has been suggested as one of the main signaling compounds to activate SAR after plant infection. I describe here the development of a matrix assisted laser desorption ionization mass spectrometry (MALDI-MS)-based approach to monitor SA in Arabidopsis thaliana. Sinapic acid was identified as the preferred choice in SA detection because minimal interference was observed between the matrix and analyte peaks. The use of Isotopic labeled Salicylic Acid (SA-d6) as a suitable internal standard for SA quantification was also demonstrated. SA spiked in plant extract at known concentrations was used as the model system to evaluate quantification and reproducibility of this newly developed MALDI-MS method. A comparison in detection feasibility between MALDI-MS and DIOS-MS was also conducted and reported in this thesis. In addition, a Liquid-phase extraction method is optimized to yield maximal extraction efficiency. I have quantified extraction efficiency and method reproducibility to extract SA in Arabidopsis leaves, and demonstrated its applicability for quantitation of endogenous SA in different plant samples. Extraction and Direct Quantification of Salicylic Acid in Arabidopsis Using Matrix- Assisted Laser Desorption Ionization Mass Spectrometry By AMEL AHMED A.GANAWI A thesis submitted to the Graduate Faculty of North Carolina State University in partial fulfillment of the requirements for the Degree of Master of Science CHEMISTRY Raleigh, NC 2007 APPROVED BY: _______________________________ Assistant Professor Lin He Chair of Advisory Committee Chemistry ________________________________ _________________________________ Professor David C. Muddiman Professor Wendy F. Boss Chemistry Plant Biology DEDICATION This thesis is dedicated to my parents, who were always there for me, for their continuous encouragement for highest levels of education, and for teaching me to use education as a measure of success. I also dedicated it to my husband for his continuous and endless support and patience. I also dedicated it to my two little sons, Ahmed and Mazin, for their time I spent away from them working on my project. ii BIOGRAPHY The author was born in Dongola, Sudan in 1970. She attended her elementary, intermediate and high school in her city of birth. After finishing her high school, her family moved to Khartoum, where she attended her undergraduate education in the Department of Chemistry at University of Khartoum. During her college life, she became more interested in pursuing higher education in chemistry. She was graduated in 1995 with Bachelor Degree in Chemistry first class honor and joined the Department of Chemistry as part timer teaching assistant. She was also nominated as a full time research assistant at The Sudan Atomic Energy Commission (SAEC), from 1995 to 1999. She was married in 1998. She moved to Saudi with her husband before she finally moved to the United States in 2002 to continue her educational journey. She joined the Department of Chemistry at North Carolina State University in the summer of 2004, where she for studied her Masters of Chemistry under supervision of Professor Lin He. iii TABLE OF CONTENTS LIST OF TABLES ................................................................................................................. vi LIST OF FIGURES .............................................................................................................. vii LIST OF SCHEMES .............................................................................................................. x CHAPTER 1: GENERAL LITERATURE OVERVIEW 1.1. Importance of Salicylic Acid (SA) as Signaling Compound..................................... 1 1.2. Current Analytical Methods for SA Detection .......................................................... 3 1.3. Overview of MALDI-MS in Small Molecule Detection ........................................... 4 1.4. References.................................................................................................................. 6 CHAPTER 2: DETECTION OF SALICYLIC ACID (SA) AS AN ESSENTIAL COMPOUND IN REGULATING SYSTEMIC DEFENSE RESPONSE IN ARABIDOPSIS 2.1. Introduction.............................................................................................................. 14 2.2. Experimental Section............................................................................................... 15 2.2.1. Material......................................................................................................... 15 2.2.2. Standard Solution Preparation ...................................................................... 16 2.2.3. Extraction Procedure..................................................................................... 16 2.2.4. MS Analysis.................................................................................................. 17 2.3. Results and Discussion ............................................................................................ 18 2.4. Conclusion ............................................................................................................... 22 2.5. References................................................................................................................ 23 CHAPTER 3: OPTIMIZATION OF SALICYLIC ACID EXTRACTION 3.1. Introduction.............................................................................................................. 37 3.2. Experimental Section............................................................................................... 38 3.2.1. Materials ....................................................................................................... 38 3.2.2. Extraction procedure..................................................................................... 39 3.2.3. Instrument and Data Analysis....................................................................... 39 3.3. Results and Discussion ............................................................................................ 40 3.4. Conclusion ............................................................................................................... 43 3.6. References................................................................................................................ 44 CHAPTER 4: AN SYSTEMATIC INVESTIGATION OF THERMAL CONTRIBUTION INSURFACE-ASSISTED LASER DESORPTION/IONIZATION MASS SPECTROMETRY (SALDI- MS) USING ORDERED NANOCAVITY ARRAYS 4.1 Introduction....................................................................................................................... 52 iv 4.2 Experimental............................................................................................................. 54 4.2.1 Materials ........................................................................................................ 54 4.2.2 Substrate preparation ..................................................................................... 55 4.2.3 Field-Emission Scanning Electron Microscope (FE-SEM) Imaging............. 56 4.2.4 Thermal Measurements.................................................................................. 56 4.2.5 Mass Spectrometry Measurements ................................................................ 57 4.3 Results and discussion .............................................................................................. 57 4.3.1Substrate Fabrication ...................................................................................... 57 4.3.2 Surface Temperature Measurements.............................................................. 59 4.3.3 Substrate Mass Spectrometry Measurements ................................................ 62 4.4 Conclusion ................................................................................................................ 63 4.5 References................................................................................................................. 65 v LIST OF TABLES CHAPTER 2: DETECTION OF SALICYLIC ACID (SA) AS AN Page ESSENTIAL COMPOUND IN REGULATING SYSTEMIC DEFENSE RESPONSE IN ARABIDOPISIS. Table 2.1. The combinatorial list of different matrices screen in SA detection 26 Table 2.2. Sample preparation of SA calibration curve 27 Table 2.3. Sample preparation table of standard SA spiked in the plant 28 extract. Table 2.4. The calculated extraction efficiency for each amount of SA added 29 to the plant tissue before the extraction. Three different independent assays were generated for each concentration to determine the inter-day precisions. CHAPTER 3: OPTIMIZATION OF SALICYLIC ACID EXTRACTION Table 3.1. Sample preparation table of standard calibration curve used to 46 calculate the amount of SA recovered after the extraction Table 3.2. Shows the change in the extraction efficiencies with respect to the 47 extraction