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Using a Combinatorial Peptide Ligand Library to Reduce the Dynamic Range of Protein Concentrations in Complicated Biological Samples

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Using a Combinatorial Peptide Ligand Library to Reduce the Dynamic Range of Protein Concentrations in Complicated Biological Samples Using a Combinatorial Peptide Ligand Library to Reduce the Dynamic Range of Protein Concentrations in Complicated Biological Samples THESIS Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in the Graduate School of The Ohio State University By Ran An Graduate Program in Chemistry The Ohio State University 2014 Master's Examination Committee: Vicki Wysocki, Advisor Dehua Pei Copyright by Ran An 2014 Abstract To improve the detection of low abundance proteins in biological fluids with proteins occurring over a wide dynamic range, a combinatorial peptide ligand library (CPLL) approach combined with two-dimensional LC- MS/MS was used. To model biomarker discovery samples, Aspergillus fumigatus proteins were spiked into patient bronchoalveolar lavage fluid (BALF). The protein profile of BALF treated with the peptide ligand library was compared with non-treated BALF. Limit of detection of Aspergillus fumigatus and recombinant catalase protein spiked separately in the BALF was evaluated. Orbitrap and Velos Pro mass spectrometric analysis showed that a BALF/bead slurry volume ratio of 20:1 (molar ratio of BALF proteins to bead capacity of 7:1) was the most ideal ratio for BALF protein concentration normalizations. In general, CPLL treated BALF lead to detection of two fold more total proteins and two fold more peptides than non-treated BALF, under the same LC-MS conditions. 60% of the total protein detected by both CPLL and non-CPLL are unique to CPLL treated samples, while 15% of the total proteins are unique to the non-CPLL sample. Preliminary data showed that the lowest spike concentrations of Aspergillus fumigatus and catalase detected in 12 μΜ BALF were 0.2 μM and 0.02 μM respectively. Preliminary data also showed that CPLL is potentially more selective towards specific proteins while removing others. ii Dedication This dissertation is dedicated to my family. iii Acknowledgments First and foremost, I would like to thank my academic advisor, Professor Vicki Wysocki, for accepting me into her group and making me appreciate the power of mass spectrometry. Without her constant contribution and practical advice, this thesis would not have been possible. A special mention for Professor Dehua, Pei, for offering insightful and detailed discussion about elution conditions for previous experiment design. It is a great pleasure to thank everyone in the Wysocki group with whom I had the honor to work. They provide a friendly and cooperative atmosphere at work. I owe sincere and earnest thankfulness to Chengsi (Michelle) Huang for being a reliable source of scientific knowledge and a caring partner throughout my graduate career. And my superwoman, Yang (Stella) Song, who is willing to come to the lab at midnight to help me trouble shoot the instrument. Thanks for all the ladies and the only gentleman, Andrew Vanschoiack, in the proteomics subgroup for helpful discussions on research progress every week. I am grateful to my colleges, Yun (Winnie) Zhang, Nilini S. Ranbaduge, Akiko Tanimoto, Matthew Bernier, Xin Ma, and Jing Yan for providing useful feedback and insightful comments on this thesis. Thanks for all the good times and the memories will be cherished forever. iv Vita 2007 to 2011 ..................................................B.S. Chemistry, Nankai University, China B.S. Chemical Engineering, Tianjin University, China 2011 to 2012 ..................................................Graduate Assistant, Department of Chemistry and Biochemistry, the University of Arizona 2012 to present ..............................................Graduate Assistant, Department of Chemistry and Biochemistry, the Ohio State University Fields of Study Major Field: Chemistry v Table of Contents Abstract…………… ........................................................................................................... ii Dedication……….. ............................................................................................................ iii Acknowledgments.............................................................................................................. iv Vita…………………. ......................................................................................................... v List of Tables…….. ........................................................................................................... ix List of Figures. .................................................................................................................... x Chapter 1. Introduction .................................................................................................. 1 1.1. Aspergillus Fumigatus Spiked in Bronchoalveolar Lavage Fluid (BALF) as Model System ................................................................................................................. 1 1.2. Establishment of Proteomics Using Mass Spectrometry ...................................... 2 1.2.1. General Workflow of Bottom-up Proteomics ....................................................... 2 1.2.2. Liquid Chromatography Coupled to Mass Spectrometry ..................................... 4 1.2.3. Electrospray Ionization and Nano Electrospray Ionization .................................. 6 1.2.4. Collision-induced Dissociation ............................................................................. 8 1.2.5. Thermo Scientific Velos Pro Mass Spectrometer ................................................. 9 1.2.6. Thermo Orbitrap Elite Mass Spectrometer ......................................................... 10 1.2.7. Database Searching ............................................................................................. 11 vi 1.3. The Quest for Low-abundance Proteins ............................................................. 12 1.3.1. Detection of Low-abundance Proteins ................................................................ 12 1.3.2. Combinatorial Peptide Ligand Library ............................................................... 12 1.3.2.1. Reduction of Protein Dynamic Concentration Range ................................... 12 1.3.2.2. Mechanism and Properties of Peptide Ligand Library in Protein Capturing 16 1.3.3. Protein Quantification in Combinatorial Peptide Ligand Library ...................... 19 Chapter 2. Study of Limit of Detection of Aspergillus fumigatus Spiked in Bronchoalveolar lavage fluid (BALF) before CPLL Treatment....................................... 21 2.1. Experimental Procedures .................................................................................... 21 2.1.1. Materials ............................................................................................................. 21 2.1.2. Bead Bed Volume Optimization for BALF ........................................................ 22 2.1.3. Antigen Spiked in BALF before CPLL .............................................................. 23 2.1.4. Trichloroacetic Acid (TCA) Precipitation .......................................................... 24 2.1.5. Protein Assay ...................................................................................................... 25 2.1.6. Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis and Silver Staining ......................................................................................................................... 25 2.1.7. Protein Digestion ................................................................................................ 26 2.1.8. LC-MS/MS ......................................................................................................... 26 2.1.9. MS Data Analysis ............................................................................................... 28 vii 2.2. Results and Discussions ...................................................................................... 29 2.2.1. Gel Results of Bed Volume Optimization for BALF ......................................... 29 2.2.2. MS Results of Bed Volume Optimization for BALF ......................................... 30 2.2.3. Gel Results for Aspergillus Fumigatus and Catalase Spiked in BALF .............. 34 2.2.4. MS Results for Aspergillus Fumigatus and Catalase Spiked in BALF .............. 36 Chapter 3. Conclusion and Future Directions .............................................................. 51 Appendix A A list of 429 BALF and Aspergillus fumigatus proteins identified on Orbitrap………. ................................................................................................................ 57 Appendix B A list of 219 BALF and catalase proteins identified on Orbitrap................. 96 Reference………….. ...................................................................................................... 109 viii List of Tables Table 2.1 Different spike concentrations and mass ratios a) Aspergillus fumigatus spiked in BALF and b) catalase spiked in BALF ......................................................................... 24 Table 2.2 Top twenty-four proteins identified from initial BALF without CPLL treatment. ........................................................................................................................................... 39 Table 2.3 Identified Aspergillus fumigatus protein groups of two BALF spiked with different amount of Aspergillus fumigatus. ...................................................................... 43 Table 2.4 Top twenty-four proteins identified from BALF without CPLL treatment. ..... 49 ix List of Figures Figure
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