A Thesis entitled Development of a HILIC-MS Approach to Quantitative Measurement of Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) by AL Mughram, Mohammed Hassan M Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Master of Science Degree in Medicinal Chemistry ______________________________________ Dr. James T Slama, Committee Chair ______________________________________ Dr. Erin G. Prestwich, Committee Member ______________________________________ Dr. Katherine A. Wall, Committee Member ______________________________________ Dr. Amanda Bryant-Friedrich, Dean College of Graduate Studies The University of Toledo August 2018 Copyright 2018, Mohammed Hassan AL Mughram This document is copyright material. Under copyright law, no parts of this document may be reproduced without the expressed permission of the author. ii An Abstract of Development of a HILIC-MS approach to quantitative measurement of nicotinic acid adenine dinucleotide phosphate (NAADP) Mohammed AL Mughram Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Master Degree in Medicinal Chemistry The University of Toledo August 2018 Nicotinic acid adenine dinucleotide phosphate, NAADP, is the most potent second messenger that triggers Ca+2 release from intracellular stores. To further understand the roles of NAADP, it will be useful to develop direct methods to measure endogenous levels of NAADP in resting cells and in addition to measure changes resulting from modulation of NAADP concentrations by stimuli. In general, the hydrophilic characteristic of nucleotides and the presence of (poly)phosphate moieties makes it difficult to separate these nucleotides under normal reversed phase conditions without using an ion-pairing agent, creating conditions that are incompatible with liquid chromatography-mass spectrometric analysis. We have successfully developed a liquid iii chromatography-mass spectrometry method without the use of ion pairing agents. We have utilized an amine functionalized stationary phase and hydrophilic interaction chromatography (HILIC) to separate and quantitate NAADP in the presence of structurally related nucleotides, including NADP, NAD, AMP, ADP, and ATP. This HILIC method is joined to a single quadrupole mass spectrometer equipped with an electrospray ion source using negative ion mode. Using this system, we can detect as little as 2 pmol of NAADP in aqueous solution and are anticipating being able to use the method to determine NAADP concentrations in mammalian cells. iv Table of Contents Abstract .............................................................................................................................. iii Table of Contents .................................................................................................................v List of Tables ..................................................................................................................... ix List of Figures ......................................................................................................................x List of Abbreviations ........................................................................................................ xii Chapter 1 Introduction .........................................................................................................1 1.1 Intracellular Ca+2 triggers ....................................................................................1 1.2 NAADP and cADPR as intracellular Ca+2 mobilizers ........................................2 1.3 Intracellular Ca+2 stores triggered by NAADP ...................................................3 1.4 Ca+2 releasing activity of NAADP ......................................................................4 1.5 NAADP Ca+2 release in Sea urchin eggs ............................................................5 1.6 NAADP Ca+2 release in mammalian cells ..........................................................6 1.6.1 NAADP releasing in acinar cells .....................................................6 1.6.2 NAADP releasing in cardiac cells ...................................................8 1.6.3 NAADP releasing in T lymphocytes ...............................................9 1.2 Assays for cellular NAADP measurement ....................................................10 v 1.2.1 Enzymatic cycling assay ................................................................................10 1.2.2 Radioreceptor binding assay ..........................................................................12 1.3 Liquid chromatography-Mass spectrometry for the analysis of cellular ……………nucleotides .....................................................................................................14 1.3.1 Mass Spectrometry-based analysis of cellular nucleotides and ... ……………dinucleotides ..................................................................................................15 1.3.2 Chromatographic separations of nucleotides .................................................17 1.3.2.1 Retention of nucleotides on the reversed-phase mode ......................18 1.3.2.2 Retention of nucleotides on HILIC mode .........................................19 1.3.3 NAADP-mass spectrometry literature review ...............................................22 1.3.4 Thesis overview .............................................................................................24 Chapter 2 Results and discussions ..................................................................................26 2.1 Procedure of synthesis of NAADP ................................................................26 2.2 Synthesis of D4-NAADP................................................................................27 2.3 HPLC development ........................................................................................30 2.4 Chromatographic separation of NAADP and other nucleotides ....................30 2.4.1 Reversed-phase separation on C18 columns........................................31 2.4.2 Reversed-phase separation on phenyl stationary columns...................32 vi 2.4.3 Chromatographic separation on HILIC columns .................................32 2.5.1 Retention studies on the various RP-columns ..............................................34 2.5.2 Retention studies on HILIC columns ............................................................37 2.5.3 Chromatographic separation of NAADP with utilization of an amine …………… stationary phase ............................................................................................39 2.6 Mass spectrometry development...................................................................42 2.6.1 Electrospray ionization .................................................................................43 2.6.2 Solvent pH effects on ESI-MS efficiency .....................................................44 2.6.3 Mass spectrometric detection of NAADP.....................................................45 2.6.4 Mass spectrometry detection.........................................................................47 2.6.5 HILIC-MS method for quantifying NAADP ................................................47 2.6.6 Spectra and observed MS/MS fragmentation patterns of NAADP ..............53 Chapter 3 Conclusion .....................................................................................................56 Chapter 4 Experimental procedures ...............................................................................59 4.1 Chromatographic methods for NAADP analyzing and separation ...............59 4.1.1 HPLC method 1 (analytical ion exchange column chromatography) ..59 4.1.2 HPLC method 2 (preparative ion exchange column chromatography).60 4.1.3 Open column method 3 (DEAE cellulose) ..........................................61 4.2 Procedure of synthesis of NAADP using the base exchange reaction ..........61 4.3 D4-NAADP synthesis....................................................................................63 vii 4.4 HPLC instrument ..........................................................................................65 4.5 HPLC separation using RP-HPLC technique ...............................................65 4.5.1 Atlantis dC18 column ..........................................................................65 4.5.2 Synergi Fusion-RP column ..................................................................66 4.5.3 Kinetex Biphenyl column ....................................................................66 4.5.4 Synergi Polar-RP column ....................................................................67 4.6 HPLC separation using HILIC technique ......................................................68 4.6.1 HILIC Kinetex column ........................................................................68 4.6.2 Luna NH2 column................................................................................68 4.7 Chemicals .......................................................................................................69 4.8 LC solvents ....................................................................................................70 4.9 LCMS-2020 instrument .................................................................................70 References ..........................................................................................................................71 Appendix ............................................................................................................................77