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Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry of Biomolecules Yu-Chen Chang Iowa State University

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Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry of Biomolecules Yu-Chen Chang Iowa State University Iowa State University Capstones, Theses and Retrospective Theses and Dissertations Dissertations 1996 Laser desorption/ionization time-of-flight mass spectrometry of biomolecules Yu-chen Chang Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/rtd Part of the Analytical Chemistry Commons Recommended Citation Chang, Yu-chen, "Laser desorption/ionization time-of-flight mass spectrometry of biomolecules " (1996). Retrospective Theses and Dissertations. 11366. https://lib.dr.iastate.edu/rtd/11366 This Dissertation is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Retrospective Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. INFORMATION TO USERS This manuscript has been rqjroduced fix>m the microfihn master. UMI fihns the text directly from the original or copy submitted. Thus, some thesis and dissertation copies are in typewriter face, w^e others may be from any type of computer printer. The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleedthrough, substandard margins, and improper alignment can adversely afifect reproduction. In the unlikely event that the author did not send IMl a complete manuscript and there are misang pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion. 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UMI A Bell & Howell Infoimation Company 300 Noith Zed) Road, Ann Aibor MI 48106-1346 USA 313/761-4700 800/521-0600 Laser desorption/ionization time-of-flight mass spectrometry of biomolecules by Yu-chen Chang A dissertation submitted to the graduate faculty in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department: Chemistry Major: Analytical Chemistry Major Professor: Edward S. Yeung Iowa State University Ames, Iowa 1996 UMI Ntimber: 9635315 UMI Microform 9635315 Copyright 1996, by UMI Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. UMI 300 North Zeeb Road Ann Arbor, MI 48103 ii Graduate College Iowa State University This is to certify that the Doctoral dissertation of Yu-chen Chang has met the dessertation reqmrements of Iowa State University Signature was redacted for privacy. Majoi^rofes Signature was redacted for privacy. For the^vlajor Department Signature was redacted for privacy. Graduate College iii To my parents iv TABLE OF CONTENTS GENERAL INTRODUCTION 1 Dissertation Organization 1 Matrix-assisted Laser Desorption/Ionization (MALDI) Time-of-flight Mass Spectrometry 1 Principle of MALDI 5 Sample preparation 6 Characteristics of MALDI 7 Quantitative analysis 8 Applications 9 Conclusions 11 Capillary Electrophoresis-Mass Spectrometry 11 Instnunentation 15 Electrospray 20 lonspray 25 Continuous-flow fast atom bombardment 26 Off-line CE-MS 30 Applications 30 Conclusions 32 V INSTRUMENTATION 35 Time-of-flight Mass Spectrometry 35 Principles 36 Mass calibration 39 Experimental set-up 39 Orthogonal extraction 40 Detection of low velocity high mass ions 45 Laser Desorption/ionization Interface for CE/MS 48 CE separation unit 48 Laser desorption/ionization interface 51 Data acquisition system 54 CHARACTERIZATION OF NEUTRAL RED AS A VISIBLE MATRIX 55 Introduction 55 Experimental 57 Sample preparation 57 TOF-MS 58 Off-line MALD and CE 58 Results and Discussions 61 Conclusions 72 vi A NEW LASER DESORPTION/IONIZATION INTERFACE FOR CAPILLARY ELECTROPHORESIS-MASS SPECTROMETRY 77 Abstract 77 Introduction 77 Experimental 79 Capillary electrophoresis 82 Laser desorption/ionization interface 82 TOF mass spectrometer 83 Data acquisition system 84 Results and Discussions 84 LDI-TOF MS 84 CE/LDI-TOF MS 92 Mechanistic study of LDI interface 96 Conclusions 101 GENERAL SUMMARY 111 REFERENCES 112 ACKNOWLEDGEMENT 121 vii LIST OF FIGURES Fig. 1 LD: irradiation of the neat analyte saunple will lead to thermal destruction. MALD: dissolving the anal3rte in an excess of matrix molecules of high spectral absorption leads to gentle desorption of analjrte molecules. 2 Fig. 2 Schematic set-up of general capillary electrophoresis. 12 Fig. 3 General illustration of a CE-MS instrumental arrangement. 16 Fig. 4 Schematic illustration of electrical connection in CE-MS interface. 18 Fig. 5 Detail of the electrospray ionization interface tip and nozzle region. 21 Fig. 6 Continuous-flow fast-atom bombardment interface for CE-MS. 27 Fig. 7 Wiley-Mclsiren dual-grid time-of-flight mass spectrometer. 37 Fig. 8 Simion plot simulation of ion trajectory. 42 Fig. 9 Schematic illustration of CE-LDI-TOF-MS which consists of capillary electrophoresis, laser desorption/ionization interface and linear time-of-flight mass spectrometer. 49 Fig. 10 LDI interface. A 15 |im timgsten wire is inserted into the CE to establish the electrical connection. 52 Fig. 11 Off-line MALD and CE set-up. 59 Fig. 12 MALDI-TOF mass spectrum of neutral red. 63 Fig. 13 MALDI-TOF mass spectrum of methionine enkephalin in excess of neutral red, the matrix to analjrte ratio is 3:1. 65 Fig. 14 MALDI-fOF mass spectra of dermenkephalin in excess of neutral red, the matrix to analyte ratio is 3:1. 67 Fig. 15 Capillary electrophoretic separation of insulin and neutral red. 70 Fig. 16 The data points indicate the ratio of the amount of insulin to NR ratio in the ablated material for different ratio of insulin to NR samples. 73 Fig. 17 The data points indicate the ratio of the amount of insulin to NR in the ablated material at different laser power density. 75 Fig. 18 Schematic illustration of CE/LDI-TOF-MS. 80 viii Fig. 19 LDI-TOF mass spectrum of (a) 0.5 mM CuClj solution and; (b) 10 nM serotonin in 0.5 mM CuCl^ solution. 85 Fig. 20 Calibration curve of serotonin obtained by using the LDI TOF MS system. 88 Fig. 21 LDI-TOF mass spectrum of 1 (iM serotonin from one laser shot. 90 Fig. 22 3D-plot of electrophoregram-mass spectrum of 1 |iM serotonin/buffer. 93 Fig. 23 Calibration curve of serotonin obtained by using the CE/LDI-TOF MS system. 97 Fig. 24 (a) Electrophoregram-mass spectrum of two-component mixture containing 10 nM serotonin and 20 ^iM tryptamine. Sample is injected by pressure flow for 5 s; (b) Total ion electrophoregram of the mixture. 99 Fig. 25 LDI-TOF mass spectrum of 50 fiM (a) Val-Leu (M.W. = 230); (b) Phe-Gly (M.W. = 222); (c) Val-Phe (M.W. = 264). 102 Fig. 26 (a) LDI-TOF mass spectrmn of 10 |iM serotonin in 0.05 mM CuClj solution; (b) LDI-TOF mass spectnmi of 10 ^iM serotonin and 1 mM nicotinic acid in 0.05 mM CuCl^ solution. 104 Fig. 27 LDI-TOF mass spectrum of (a) 50 |liM Val-Leu; (b) 50 |iM Val-Leu plus 20 mM nicotinic acid; (c) 20 mM nicotinic acid. 106 Fig. 28 LDI-TOF mass spectrum of (a) 50 |iM Val-Leu; (b) 50 |iM Val-Leu plus 20 mM 2,5-dihydroxybenzoic acid; (c) 20 mM 2,5-dihydroxybenzoic acid. 108 ix LIST OF TABLES Table I. The most commonly used matrices for MALDI MS. 4 Table II. Detection modes developed for capillary electrophoresis. 14 Table III. Performance characteristics of mass spectrometers for CE/MS. 33 Table IV. Reproducibility of capillary electrophoresis-mass spectrometry for 1 |iM serotonin. 95 1 GENERAL INTRODUCTION Desertation Organization This dissertation begins with the general introduction of literaure related to this work, including the background concepts and recent progress in this area. The following chapters are the research work of the author and they are followed by a general summary. The chapter proceeding the general summary is a journal paper to be published. A list of all the references cited concludes this dissertation. Matrix-assisted Laser Desorption/Ionization (MALDI) Time-of-flight Mass Spectrometry Since 1970 lasers have been used to generate ions for analysis of organic molecules in mass spectrometers (1). Molecules are ejected from a solid surface following irradiation with a pulsed laser beam. By this way, it is possible to transfer involatile species from the solid to the gas phase within a mass spectrometer, allowing ionization and mass analysis to take place. Biomolecules normally present in the condensed phase need to be converted into intact, isolated ionized molecules in the gas phase for MS analysis. This conversion is diflBcult to achieve because biomolecules are polar and massive, and therefore extremely involatile. Laser desorption provides a good solution for biomolecule analysis. However, there is a upper limit to the size of molecules that could be desorbed as intact ions. This limit, which depends on molecular structure and laser parameters, is near a mass of 1000 Da for biopoljmaers and up to 9000 Da for S3nithetic polymers (2). The main breakthrough toward higher masses came when the Hillenkamp and Karas research group realized that the use of a matrix could 2 circumvent this problem (3). A matrix is a small and highly absorbing species, such as nicotinic acid. A low concentration of analyte molecules are embedded in either a solid or a liquid matrix (Fig.
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