MASS SPECTROMETRY-BASED INVESTIGATIONS TO CHARACTERIZE SPECIFIC CELL TYPES WITHIN THE BRAIN BY ANN M. KNOLHOFF DISSERTATION Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Chemistry in the Graduate College of the University of Illinois at Urbana-Champaign, 2011 Urbana, Illinois Doctoral Committee: Professor Jonathan V. Sweedler, Chair Assistant Professor Ryan C. Bailey Professor Martha U. Gillette Professor Alexander Scheeline ABSTRACT Investigations of the chemical content of the brain and its many constitutive cell types yield information regarding normal and abnormal brain function. Frequently, proteomics, peptidomics, and metabolomics experiments survey brain regions and attribute detected species to neurons. However, many other cell types are present, including astrocytes, oligodendrocytes, and microglia. It is also known that different cell types can vary greatly in their analyte content and concentration; therefore, to obtain an adequate representation of brain function, specific cell types require further characterization. Cell types of particular interest are astrocytes, which are involved in neuronal communication, and mast cells, which are involved in allergic response. Moreover, morphologically similar cells can exhibit chemical heterogeneity; to characterize these differences, single-cell analyses are necessary. In this case, single cells from the model organism, Aplysia californica, are isolated for analysis. Mass spectrometry (MS) is well-suited for such applications because it has limits of detection in the attomole range for both small molecule metabolites and peptides, has a wide dynamic concentration range, and can detect and identify molecules of interest without a priori knowledge of sample content. To characterize metabolite profiles in single cells and tissue homogenates, a laboratory-built capillary electrophoresis (CE) system coupled to electrospray ionization (ESI) MS is implemented. This is a useful technique for such measurements because it only requires a small amount of sample (injection of nanoliter volumes), it can efficiently separate and detect hundreds of analytes within a single CE separation, and it has detection limits in the low nanomolar (attomole) range for several neurotransmitters. This technique has been ii successfully applied to characterize and chemically distinguish single cells of various neuron types from Aplysia californica. Here, hundreds of ions are detected within single-cell samples and 36 ions are identified. Furthermore, neuron-specific analytes are revealed, chemical classification of neurons is achieved via principal component analysis, and cellular concentrations of serine and glutamic acid are determined for the different neuron types. CE- ESI-MS has also been effective in determining relative chemical differences between different sample types. To determine the chemical contribution that mast cells make to the brain, tissue homogenates from mast cell-deficient mice are compared to their heterozygous littermates. In this comparison, a number of metabolites, including amino acids and choline, are statistically different between the two sample types; furthermore, this data agrees with differences observed in gene expression data. The combined metabolite and transcriptomics data reveal global chemical differences that affect a number of metabolic pathways, which may be related to behavioral and developmental traits observed in mast cell-deficient mice. The characterization of peptide content in astrocytes is also achieved by employing several instrumental platforms, such as CE-ESI-MS and matrix-assisted laser desorption/ionization MS. By carefully selecting defined samples, the identification of a large number of peptides from purified astrocyte samples is accomplished. iii ACKNOWLEDGMENTS There are a number of people that I would like to thank who contributed to the success of this work. First of all, I would like to thank my advisor, Prof. Jonathan Sweedler, for his continual guidance and encouragement throughout my graduate career. His mentoring style, approach to problem solving, and ability to see the big picture have helped me to become an independent and better scientist. I would also like to thank the members of my committee, Profs. Ryan Bailey, Martha Gillette, and Alexander Scheeline, for their encouragement and support. I would also like to thank Prof. Bill Greenough, who originally served on my preliminary exam committee, for his initial input regarding this work. I have had the wonderful opportunity to work with a number of collaborators on interesting projects. I would like to thank Larry Millet from Prof. Martha Gillette’s lab for discussing research ideas, providing samples, and initial training with respect to cell culture. Harry Rosenberg, also from Prof. Martha Gillette’s lab, provided me with many samples to test and was always willing to try new research avenues. I also had the opportunity to collaborate with Prof. Rae Silver and Kate Nautiyal from Columbia University and Sergey Kalachikov and Irina Morozova from the Columbia Genome Center. I very much appreciate the exchange of ideas and effort put forth by this group of people. It has been a privilege to work in the Sweedler research group. Both past and present group members have been willing to lend a hand when needed and to discuss different ways of approaching challenges that arise. In particular, I would like to thank Michael Heien who provided my initial training when I joined the group, Ping Yin for collaborative efforts and discussions, Ted Lapainis for original training on the CE-ESI-MS instrument platform, Peter iv Nemes for collaborative efforts in CE-ESI-MS investigations, and Stanislav Rubakhin for cell isolations and helpful discussions. Additionally, multiple people have helped on various aspects of the performed research and provided general advice; this list includes Christine Cecala, Chris Dailey, Xiaowen Hou, Jamie Iannacone, Zhen Li, Eric Monroe, Nobutoshi Ota, Elena Romanova, Ting Shi, and Fang Xie. I would also like to thank Stephanie Baker for her efforts in finalizing manuscripts prior to publication. I also gratefully acknowledge Julie Sides, who is always willing to help graduate students with anything they need. Finally, I would like to thank my friends and family. Their continual friendship and support is very much appreciated. v TABLE OF CONTENTS CHAPTER 1. INTRODUCTION ..............................................................................................1 1.1 Research Motivation ........................................................................................................1 1.2 Thesis and Research Overview ........................................................................................2 1.3 Cell Types and Model Systems ........................................................................................3 1.3.1 Aplysia californica.....................................................................................................3 1.3.1.1 Aplysia Neurons .................................................................................................4 1.3.2 Mus musculus and Rattus norvegicus ........................................................................5 1.3.2.1 Mast Cells .........................................................................................................6 1.3.2.2 Astrocytes .........................................................................................................7 1.4 Conclusions ......................................................................................................................9 1.5 Tables .............................................................................................................................10 1.6 References .....................................................................................................................11 CHAPTER 2. SINGLE-CELL MASS SPECTROMETRY ....................................................16 2.1 Introduction ....................................................................................................................16 2.2 Mass Spectrometry .........................................................................................................18 2.2.1 Matrix-Assisted Laser Desorption/Ionization .........................................................19 2.2.1.1 Sample Preparation for Single-Cell MALDI ...................................................21 2.2.1.2 Recent Applications of Single-Cell MALDI ...................................................23 2.2.2 Secondary Ion Mass Spectrometry ..........................................................................27 2.2.2.1 Sample Preparation for Single-Cell SIMS .......................................................28 2.2.2.2 Recent Applications of Single-Cell SIMS .......................................................29 vi 2.2.3 Electrospray Ionization ............................................................................................33 2.2.3.1 Recent Applications of Single-Cell ESI ..........................................................34 2.2.4 Other MS Approaches .............................................................................................35 2.3 Overall Outlook for Single-Cell MS ..............................................................................36 2.4 Figures ............................................................................................................................37