UCLA UCLA Electronic Theses and Dissertations Title Developing Top-Down Mass Spectrometry for Intact Protein Identification in the Chromatographic Timescale Permalink https://escholarship.org/uc/item/16f1599t Author Lakshmanan, Rajeswari Publication Date 2012 Peer reviewed|Thesis/dissertation eScholarship.org Powered by the California Digital Library University of California UNIVERSITY OF CALIFORNIA Los Angeles Developing Top-Down Mass Spectrometry for Intact Protein Identification in the Chromatographic Timescale A dissertation submitted in partial satisfaction of the requirements for the degree Doctor of Philosophy in Biochemistry and Molecular Biology by Rajeswari Lakshmanan 2012 ABSTRACT OF THE DISSERTATION Developing Top-Down Mass Spectrometry for Intact Protein Identification in the Chromatographic Timescale by Rajeswari Lakshmanan Doctor of Philosophy in Biochemistry and Molecular Biology University of California, Los Angeles, 2012 Professor Joseph A. Loo, Chair Protein identification by top-down mass spectrometry based methods yield intact mass of the proteins and indicate the presence of post-translational modifications (PTMs) and/or isoforms. Currently, the methods employed for top-down protein identification are performed using instruments with dual mass analyzers and are based on fragmenting isolated charge states, which greatly reduces the duty cycle of the instrument. High throughput top-down methods are required for protein identification in complex sample mixtures. We demonstrate the capability to perform intact protein identifications in a single-stage time-of-flight mass spectrometer during protein elution from a liquid chromatography (LC) column. In addition, we have developed a new data-independent fragmentation method known as ‘Continuous Accumulation of Selected Ions– Collisionally Activated Dissociation’ (CASI-CAD) to fragment multiple charge states of the protein simultaneously for the purpose of identification in the LC timescale. CASI-CAD is performed without any precursor selection and thus, the duty-cycle of the instrument is not ii lowered. Both these methods unambiguously identified all the proteins in the human proteasome complex used for method development. The presence of PTMs and N-terminal modifications were also characterized for the proteins in this complex. Supercharging reagents are known for their ability to enhance the multiple charging of proteins during electrospray ionization (ESI). This improves the mass measurement accuracy and fragmentation efficiency of proteins during ESI-MS. Currently, the mechanism behind supercharging is unknown. We have analyzed different supercharging reagents under a variety of solvent conditions to probe the mechanisms behind supercharging. In addition, the supercharging ability of sulfolane was utilized for proteins eluting from a column by adding the reagent to the LC solvents. Furthermore, reagent introduction in the vapor phase increased the signal intensity for intact proteins eluting from a column when compared to experiments performed without the reagent. These methods presented here are efficient top-down means to address complex samples in the chromatographic timescale. iii The dissertation of Rajeswari Lakshmanan is approved. Catherine F. Clarke Julian P. Whitelegge Steven G. Clarke Thomas G. Graeber Joseph A. Loo, Committee Chair University of California, Los Angeles 2012 iv DEDICATION To my wonderful parents and loving husband v TABLE OF CONTENTS LIST OF FIGURES ................................................................................................................ xi LIST OF TABLES................................................................................................................. xiv LIST OF SUPPLEMENTAL FIGURES ................................................................................... xv ACKNOWLEDGMENTS ....................................................................................................... xvi VITA ..................................................................................................................................... xx CHAPTER 1: Introduction ....................................................................................................... 1 1.1. Mass spectrometry based proteomics ......................................................................... 1 1.2. Top-Down and Bottom-Up MS .................................................................................... 1 1.3. Protein identification and characterization by TD ......................................................... 3 1.4. A short chronological history on the development of TD methods................................. 4 1.5. Mass analyzers for TD proteomics .............................................................................. 6 1.5.1. FT-ICR MS ........................................................................................................ 6 1.5.2. Orbitrap............................................................................................................. 8 1.5.3. Quadrupole time-of-flight ................................................................................... 9 1.5.4. Ion trap ........................................................................................................... 12 1.6. Intact protein fragmentation schemes ....................................................................... 13 1.6.1. Collisionally activated dissociation .................................................................... 13 1.6.2. Higher energy collision dissociation .................................................................. 14 1.6.3. Electron capture dissociation ........................................................................... 14 1.6.4. Electron transfer dissociation ........................................................................... 15 1.6.5. Other miscellaneous fragmentation methods .................................................... 17 1.7. Intact protein separation techniques ......................................................................... 17 1.7.1. Electrophoresis ............................................................................................... 17 vi 1.7.1.1. Gel-based techniques ............................................................................. 17 1.7.1.2. Isoelectric focusing.................................................................................. 19 1.7.1.3. Capillary electrophoresis ......................................................................... 20 1.7.2. Liquid chromatography .................................................................................... 21 1.7.2.1. Reversed phase chromatography ............................................................ 21 1.7.2.2. Ion-exchange chromatography ................................................................ 22 1.7.2.3. Size-exclusion chromatography ............................................................... 22 1.7.2.4. Multi-dimensional separation ................................................................... 22 1.8. TD data analyses ..................................................................................................... 23 1.8.1. ProSight .......................................................................................................... 24 1.8.2. BIG Mascot ..................................................................................................... 25 1.8.3. MS-Align+ ....................................................................................................... 25 1.8.4. Precursor ion independent top-down algorithm ................................................. 25 1.9. Supercharging ......................................................................................................... 26 1.10. Current limitations and future directions .................................................................. 32 1.11. Summary ............................................................................................................... 35 1.12. References ............................................................................................................ 37 CHAPTER 2: Top-Down Protein Identification using a Single-Stage LC-ESI-TOF Mass Spectrometer .................................................................................................. 52 2.1. Abstract ................................................................................................................... 52 2.2. Introduction ............................................................................................................. 53 2.3. Experimental section ................................................................................................ 56 2.3.1. Sample preparation ......................................................................................... 56 2.3.2. LC conditions .................................................................................................. 57 2.3.3. MS parameters................................................................................................ 57 2.3.4. Data analysis .................................................................................................. 58 vii 2.4. Results and discussion ............................................................................................ 58 2.4.1. Optimization of fragmentor voltage ................................................................... 58 2.4.2. LC-MS of standard protein mix