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Characterization of the Extracellular Proteome of a Natural Microbial Community with an Integrated Mass Spectrometric / Bioinformatic Approach

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Characterization of the Extracellular Proteome of a Natural Microbial Community with an Integrated Mass Spectrometric / Bioinformatic Approach University of Tennessee, Knoxville TRACE: Tennessee Research and Creative Exchange Doctoral Dissertations Graduate School 12-2010 Characterization of the Extracellular Proteome of a Natural Microbial Community with an Integrated Mass Spectrometric / Bioinformatic Approach Brian Keith Erickson UTK, [email protected] Follow this and additional works at: https://trace.tennessee.edu/utk_graddiss Part of the Bioinformatics Commons, Environmental Microbiology and Microbial Ecology Commons, and the Systems Biology Commons Recommended Citation Erickson, Brian Keith, "Characterization of the Extracellular Proteome of a Natural Microbial Community with an Integrated Mass Spectrometric / Bioinformatic Approach. " PhD diss., University of Tennessee, 2010. https://trace.tennessee.edu/utk_graddiss/879 This Dissertation is brought to you for free and open access by the Graduate School at TRACE: Tennessee Research and Creative Exchange. It has been accepted for inclusion in Doctoral Dissertations by an authorized administrator of TRACE: Tennessee Research and Creative Exchange. For more information, please contact [email protected]. To the Graduate Council: I am submitting herewith a dissertation written by Brian Keith Erickson entitled "Characterization of the Extracellular Proteome of a Natural Microbial Community with an Integrated Mass Spectrometric / Bioinformatic Approach." I have examined the final electronic copy of this dissertation for form and content and recommend that it be accepted in partial fulfillment of the requirements for the degree of Doctor of Philosophy, with a major in Life Sciences. Robert L. Hettich, Major Professor We have read this dissertation and recommend its acceptance: Alison Buchan, Loren J. Hauser, Cynthia B. Peterson, Brynn H. Voy Accepted for the Council: Carolyn R. Hodges Vice Provost and Dean of the Graduate School (Original signatures are on file with official studentecor r ds.) To the Graduate Council: I am submitting herewith a dissertation written by Brian Keith Erickson entitled “Characterization of the Extracellular Proteome of a Natural Microbial Community with an Integrated Mass Spectrometric / Bioinformatic Approach.” I have examined the final electronic copy of this dissertation for form and content and recommend that it be accepted in partial fulfillment of the requirements for the degree of Doctor of Philosophy, with a major in Life Sciences. Robert Hettich , Major Professor We have read this dissertation and recommend its acceptance: Alison Buchan Loren Hauser Cynthia Peterson Brynn Voy Accepted for the Council: Carolyn R. Hodges Vice Provost and Dean of the Graduate School (Original signatures are on file with official student records.) Characterization of the Extracellular Proteome of a Natural Microbial Community with an Integrated Mass Spectrometric / Bioinformatic Approach A Dissertation Presented for the Doctor of Philosophy Degree The University of Tennessee, Knoxville Brian Keith Erickson December 2010 Copyright © 2010 by Brian Keith Erickson All rights reserved. ii DEDICATION I dedicate this dissertation to my family, especially my wife, Alison Russell Erickson. Their support, encouragement and love has shaped who I am today and without them I would not have had the opportunities that have preceded this dissertation. iii ACKNOWLEDGEMENTS I would like to thank my advisor, Dr. Robert Hettich for his support and consistent, thoughtful, and embedded approach throughout the course of this work. I would like to acknowledge my committee members: Dr. Alison Buchan, Dr. Loren Hauser, Dr. Cynthia Peterson, and Dr. Brynn Voy for their time, excellent comments, suggestions, and thought provoking discussions. I would like to thank the Organic and Biological Mass Spectrometry group at Oak Ridge National Laboratory for their patience and guidance. I would also like to thank the staff, Dr. Cynthia Peterson and Dr. Albrecht von Arnim of the Genome Science and Technology program at the University of Tennessee for the professional enrichment and personal support. I would like to thank my wife, Alison, and close friends, (Dr.) Scott Younger and Dr. Rich Giannone, for their invaluable friendship and support. Finally, I would like to thank my parents, Jim and Jennifer; and Jeff and Gretchen for their love and support. iv ABSTRACT Proteomics comprises the identification and characterization of the complete suite of expressed proteins in a given cell, organism or community. The coupling of high performance liquid chromatography (LC) with high throughput mass spectrometry (MS) has provided the foundation for current proteomic progression. The transition from proteomic analysis of a single cultivated microbe to that of natural microbial assemblages has required significant advancement in technology and has provided greater biological understanding of microbial community diversity and function. To enhance the capabilities of a mass spectrometric based proteomic analysis, an integrated approach combining bioinformatics with analytical preparations and experimental data collection was developed and applied. This has resulted in a deep characterization of the extracellular fraction of a community of microbes thriving in an acid mine drainage system. Among the notable features of this relatively low complexity community, they exist in a solution that is highly acidic (pH < 1) and hot (temperature > 40°C), with molar concentrations of metals. The extracellular fraction is of particular interest due to the potential to identify and characterize novel proteins that are critical for survival and interactions with the harsh environment. The following analyses have resulted in the specific identification and characterization of novel extracellular proteins. In order to more accurately identify which proteins are present in the extracellular space, a combined computational prediction and experimental identification of the extracellular fraction was performed. Among the hundreds of proteins identified, a highly abundant novel cytochrome was targeted and ultimately characterized through high performance MS. In order to achieve deep proteomic coverage of the extracellular fraction, a metal affinity based protein enrichment utilizing seven different metals was developed and employed resulting in novel protein identifications. A combined top down and bottom up analysis resulted in the characterization of the intact molecular forms of extracellular proteins, including the identification of post-translational modifications. Finally, in order to determine the effectiveness of current MS methodologies, a software package was designed to characterize the > 100,000 mass spectra collected during an MS v experiment, revealing that specific optimizations in the LC, MS and protein sequence database have a significant impact on proteomic depth. vi TABLE OF CONTENTS Chapter 1: Introduction to Proteome Mass Spectrometry and Rationale for Characterization of an Extracellular Fraction of a Natural Microbial Community………...1 Chapter 2: An Integrated Experimental / Computational MS Based Platform for the Proteomic Characterization of a Natural Microbial Community…………………………..16 Chapter 3: Computational Prediction and Experimental Validation of Signal Peptide Cleavages in the Extracellular Proteome of a Natural Microbial Community………………………………..32 Chapter 4: An Integrated, Comparative Metal Affinity Enrichment and Proteomic Characterization of Novel Proteins from a Natural Microbial Community………………………………………58 Chapter 5: High Resolution Mass Spectrometry for the Characterization of a Novel, Growth Stage Dependent Cytochrome……………………………………………………...84 Chapter 6: The Design and Implementation of Software for Integrating Bottom up and Top Down MS Datasets for the Characterization of an Extracellular Fraction from a Natural Microbial Community…………………………………………..…………....96 Chapter 7: Development of a Spectral Assignment Approach to Evaluate Assigned versus Unassigned Tandem Mass Spectra in the Proteomic Analyses of Microbial Isolates and Communities……………..........................................................................117 Chapter 8: Conclusions and Significance of the Characterization of the Extracellular Fraction in a Natural Microbial Community………………………………………….........143 References…………………………………………………………………………….….....150 Vita………………………………………………………………………………………….....161 vii LIST OF TABLES 3.1: Summary of Computational Prediction and MS Identification of Signal Peptide Cleaved Proteins from 5 Distinct Extracellular AMD Samples………………………41 3.2: Distribution of Predicted Signal Peptide Cleaved Proteins for the Dominant Microbes in the AMD Microbial Community…………………………………………….42 3.3: Results of Edman N-terminal Sequencing of Select Secretome Proteins from the AMD Microbial Community…………………………………………………………….46 3.4: Highly Conserved and Replicated Signal Peptide Cleaved Proteins with Confirming N-terminus Spectra……………………………………………………………49 3.5: NSAF Comparison of Select, Highly Differential Signal Peptide Cleaved Proteins………………………………………………………………………………………...51 3.6: Pfam Domain Analysis of Conserved and High Confidence Signal Peptide Cleaved Proteins……………………………………………………………………......……53 4.1: Number of Proteins Labeled as Uniquely IMAC Bound or Unbound After Analysis of MS Data from Chromatographic Fractions…………………………….…68 4.2: Average Amino Acid Abundance within Protein Groups that are Bound to Specific Metals, or Groups of Metals…………………………………………….…........71 4.3: Identified Flagellar Proteins and Their Pattern of IMAC Enrichment…...…….75 4.4: Pfam Identifications
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