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U UNIVERSITY OF CINCINNATI Date: I, , hereby submit this original work as part of the requirements for the degree of: in It is entitled: Student Signature: This work and its defense approved by: Committee Chair: Approval of the electronic document: I have reviewed the Thesis/Dissertation in its final electronic format and certify that it is an accurate copy of the document reviewed and approved by the committee. Committee Chair signature: Investigation of Phosphorylated Proteins and Peptides in Human Cerebrospinal Fluid via High-Performance Liquid Chromatography Coupled to Elemental and Molecular Mass Spectrometry A thesis submitted to the Graduate School of the University of Cincinnati In partial fulfillment of the Requirements for the degree of MASTER OF SCIENCE In the Department of Chemistry of the College of Arts and Sciences By ORVILLE DEAN STUART B.S., Chemistry The University of Texas at Tyler, Tyler, Texas May 2006 Committee Chair: Joseph A. Caruso, Ph.D Abstract Cerebrospinal fluid (CSF) surrounds and serves as a protective media for the brain and central nervous system (CNS). This fluid remains isolated from other biological matrices in normal bodily conditions, therefore, an in depth analysis of CSF has the potential to reveal important details and malfunctions of many diseases that plague the nervous system. Because phosphorylation of a wide variety of proteins governs the activity of biological enzymes and systems, a method for the detection of 31P in proteins found in human cerebrospinal fluid by high-performance liquid chromatography (HPLC) coupled to inductively coupled plasma mass spectrometry (ICPMS) is described. Specifically, it is of interest to compare phosphorylated proteins/peptides from patients suffering from post subarachnoid hemorrhage (SAH) arterial vasospasms against CSF from non-diseased patients. HPLC provides a way to separate many of the components of the CSF matrix, while ICPMS allows for the simultaneous detection of various elements, specifically phosphorus, at high sensitivity. Because structural elucidation is lost in the ICPMS experiment, softer ionization techniques such as Electrospray Ionization (ESI) are thus utilized for potential protein/peptide identification. Database searching software (Spectrum Mill) is then used in this study as a means of identification and validation. SEC- ICPMS confirms the presence of phosphorylated peptides within normal CSF as well as vasospastic and non-vasospastic SAH CSF samples. Non-enzymatically digested samples with ESI-MS analysis, in conjunction with Spectrum Mill database searching, indicates a variance among phosphorylated protein species across the sample batches, however, no correlations are drawn between disease types and phosphoprotein presence or absence. iii © Copyright MMIX Orville Dean Stuart All Rights Reserved iv Preface and Acknowledgements To this day, I am often reminded of an artfully written piece of prose by Sully-Prudhomme† which, in my opinion, glamorized the daily life of a Chemist: “Surrounded by beakers, by strange coils, By ovens and flasks with twisted necks, The chemist, fathoming the whims of attractions, Artfully imposes on them their precise meetings. He controls their loves, hidden until now, Discovers and directs their secret affinities, Unites them and brings about their abrupt divorces, And purposefully guides their blind destinies.” For me, just knowing that in the grand scheme of things, I could one day be able to “guide the destiny” of some unseen object made me shiver with excitement. I suppose that’s why I decided to become a Chemist at a young age. Such a decision, I’ve realized over the years, doesn’t simply come just as a whim. Without a doubt, as a Christian, I am in constant awe of God’s wondrous “quantum” world; literally the world of the unseen. As a Scientist, I am humbled by the fact that God has seen fit to open one of the million windows to his wonders, and has allowed me to make a report about it. Of course, I must also thank my father, Dr. John G. Stuart for his ever watchful, ever thoughtful guidance throughout my chemistry career, as well as my mother, Joyce Stuart, and my siblings Holly and Jonathan for always being supportive of my, to borrow the phrase, “weird science-y stuff.” Unlike my Organic father, I decided to go down the Analytical route; so much for “guiding blind destinies.” Or so I thought, before I ventured into the world of ICPMS. Artfully breaking down molecules and compounds and ionizing them in a PLASMA of all things… “too v cool!” I would think every day as I sat in front of the instrument. Being a Sci-Fi buff, something of this nature truly seemed to have the same scientific “cool factor” as Star Trek, or Mobile Suit Gundam. It therefore goes without saying, that Dr. Joseph Caruso made this all possible. Through him, I was able to explore a childhood dream of not only “guiding the destiny” of unseen analytes, but he also allowed me to see first hand the rigors of Ph.D life through a different lens than that of my father. Throughout my graduate school and ICPMS career, the one person who constantly challenged me, both academically and personally, was Jennifer Siverling. Ever the amazing classmate, labmate, and more importantly, love of my life, Jenn without a doubt changed my life forever for the better. Every moment was a rip-roaring ride of fun, love, excitement, and lots of late nights in the Lab, slaving over instrumentation malfunctions and bad data! Even through the good and the bad, we stuck together strong… And before you ask, YES I find it something of a personal triumph that I managed to get you to like anime… well… some anime! You were right when you said it was all like an up and down rollercoaster… but you know what? That’s what made it special; it’s what made us “us,” and I would never trade a moment for the world! Well… with all that said, I suppose it’s time to get this thesis started! To old friends lost, and to new ones not fully realized…; in the same cryptic vein as Yoshiyuki Tomino‡, this one’s for you… vi “And now… In anticipation of your Insight into the future…” vii Table of Contents Abstract…………………………………………………………………………………………………………………………………… iii Preface and Acknowledgements..……………………………………………………………………………..……………… v List of Tables……………………………………………………………………………………………………………………….…… ix List of Figures………………………………………………………………………………………………………………………….… x Introduction…………………………………………………………………………………………………………………………….. 1 Cerebrospinal Fluid………………………………………………………………………………………………………. 2 Instrumental Overview…………………………………………………………………………………………………………….. 3 Inductively Coupled Plasma Mass Spectrometry………………………………………………………….. 3 Size Exclusion Chromatography…………………………………………………………………………….……… 5 Electrospray Mass Spectrometry……………………………………………………………………………..…… 6 Experimental……………………………………………………………………………………………………………………….…… 8 Results………………………………………………………………………………………………………………………………..….. 11 Chromatographic and Spectroscopic Data Analysis……………………………………………….….… 11 Protein Identification Discussion……………………………………………………………………………...… 23 Closing Remarks/Future Studies………………………………………………………………………………………..…… 28 References……………………………………………………………………………………………………………………….….… 30 Appendix 1……………………………………………………………………………………………………………………….……. 32 viii List of Tables Table Page 1. CSF sample nomenclature, disease type, and mass-volume conversion factor……………………… 9 2. Instrumental parameters………………………………………………………………………………………………….… 10 ix List of Figures Figure Page 1. Step by step diagram of microfluidic re-routing within the nanoLC Electrospray Chip………..… 7 2. 31P counts per second of “normal” control non-SAH CSF 109-Con sample………….……….……… 11 3. 31P counts per second of non-vasospastic SAH CSF samples…………………….…………………….…… 11 4. 31P counts per second of vasospastic SAH CSF samples…………………………………….………………… 12 5. Overlayed ESI BPCs of “normal” control non-SAH CSF (109-Con) and SEC-HPLC trace..………. 14 6. Overlayed ESI BPCs of non-vasospastic SAH CSF (NV 71-01) and SEC-HPLC trace……….………. 15 7. Overlayed ESI BPCs of non-vasospastic SAH CSF (NV 111-1) and SEC-HPLC trace…….…………. 16 8. Overlayed ESI BPCs of non-vasospastic SAH CSF (NV 113-1) and SEC-HPLC trace….……………. 17 9. Overlayed ESI BPCs of vasospastic SAH CSF (V 108-2) and SEC-HPLC trace…….…………………… 18 10. Overlayed ESI BPCs of vasospastic SAH CSF (V 109-2) and SEC-HPLC trace…….…………………. 19 11. Overlayed ESI BPCs of vasospastic SAH CSF (V 112-1) and SEC-HPLC trace…….…………………. 20 x Introduction The phosphorylation and dephosphorylation of both structural and regulatory proteins as post translational modifications (PTMs) are a major intracellular control mechanism present in eukaryotes [1]. With the addition of phosphate to a specific activation site, an otherwise inactive protein, or enzyme as a collective whole, may change shape due to electrostatic repulsions and interactions, adopting a new “active” conformation, wherein the protein or enzyme is prepared to perform specific biological functions [2]. When biological systems are disrupted due to outside physical trauma or internal diseases, the various ways of how regulatory proteins are spurred into action, or the resulting loss of action due to variations in phosphorylated proteins and or peptides, becomes an interesting topic to investigate and may push the discovery of biomarkers to indicate and ultimately suggest preventive measures. The controls and regulations involved within the central nervous system (CNS) are of particular interest because it is the control center of the human body. Indeed, much work has