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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

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

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…

“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

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

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 been done on the study of degenerative neurological disorders such as Alzheimer’s disease, convulsions, and ischemic stroke as well as physical head injuries [3,4,5,6] with the final goal of suggesting a correlation between a condition with a corresponding rise or fall in metal/metalloid concentration within a patient’s cerebrospinal fluid (CSF). CSF is a fluid secreted from the choroid plexus which surrounds the CNS providing both buoyancy and protection [7] and contains various salts, enzymes, proteins, peptides and other small metabolites, all of which are key for regulated and normal physiological processes [8].

Phosphorylated proteins and peptides in CSF have been showcased by Yuan and

Desiderio in 2005 by way of conventional proteomics approaches [9] and most recently by Ellis

1 et al. in 2008 through the use of capillary HPLC (capHPLC) coupled with ICPMS for elemental specific 31P screening of CSF samples and nano HPLC coupled with ESI-MS for protein identification [10]. Element specific spectroscopy techniques, namely ICPMS, have seen a growth in its use for analyzing phosphorus and phosphorylation in both proteins and peptides

[11,12,13,14,15,16,17,18] and is strong proof of the robustness and high sensitivity of ICPMS when coupled to HPLC, most frequently in the lower solvent consumption form of capillary

HPLC, as a means to segregate and create uniform sample introduction into the ionizing plasma. In conjunction with the use of a collision/reaction cell, ICPMS is able to discriminate against multiple polyatomic interferences for 31P in particular, (14N16O1H+, 15N15N1H+, 15N16O+,

14N17O+, 13C18O+, 12C18O1H+) [19,20] thus increasing the detection capabilities.

Cerebrospinal Fluid

In this study, CSF from patients afflicted by subarachnoid hemorrhage (SAH) is probed for phosphorylated proteins and or peptides in two cases: samples of SAH CSF from patients who suffered from post hemorrhagic vasospasm and those who did not. Cadoux-Hudson, Pyne, and

Clark describe eloquently the vasospasm and it’s onset [21] as being the sudden constriction of blood vessels surrounding the site of SAH due to the breakdown of blood products now in direct contact with the brain and the subarachnoid space. This constriction then effectively kills the affected portions of the brain, ultimately causing patient death. Because these breakdown products are now in contact with the fluid surrounding the subarachnoid space, it is of interest to examine patient CSF with the goal of noting variations and differences in phosphorylated proteins and or peptides across the two disease types, and also to compare

2 phosphoproteins/peptides found in normal CSF from a healthy patient for a more rounded comparison. Differences in phosphorylation may suggest the discovery of biomarkers for vasospasm and SAH. Specifically, this study focuses on the analysis of peptides and proteins found within this matrix, which are less than 5 kDa in molecular weight, thereby omitting any abundant higher molecular weight proteins present, such as albumin and immunoglobulins, which may mask the presence of smaller moieties during chromatographic screening.

Instrumental Overview

In performing this research, chromatographic as well as mass spectrometric techniques were utilized, namely size exclusion chromatography in order to segregate sample components by molecular weight (hydrodynamic radii), and ICPMS as well as ESI-MS for the detection of phosphorus containing protein moieties and structural analysis, respectively.

Inductively Coupled Plasma Mass Spectrometry

The “grounding” point of this research revolved around the use of the inductively coupled plasma mass spectrometer experiment. There exist countless applications of ICPMS for a variety of analytical purposes. This element specific MS technique allows for the detection of specific element containing species within a suitable sample matrix (in this case, phosphorus containing peptides and or proteins within human cerebrospinal fluid). The aqueous sample is first introduced into a nebulizer to produce a fine aerosol. Past the nebulizer, the spray chamber serves as a means to desolvate and direct the aerosol towards the plasma torch center, as well as to remove any large aerosol particles, which otherwise would not undergo

3 ionization in the plasma. The plasma is generated by a radio frequency (RF) signal which is passed along a water-cooled copper coil about the tip of the torch, which itself is comprised of a set of three quartz concentric tubes through which argon gas flows. This RF signal generates a magnetic field that couples to the flowing analyte stream. A spark creates argon ions, argon metastables, and electrons to create an argon plasma (with temperatures reaching 7000K). The argon ions are induced into a circular motion which results in the iconic annular plasma.

Sustaining the plasma is done by maintaining both the RF induced magnetic field and a steady stream of argon gas through the torch system. The fine aerosol of sample is then passed through the center of this plasma annulus where it is then desolvated and ionized.

After ionization, a series of cones and focusing elements direct the ionized sample into a collision/reaction cell, which is comprised of an octopole encased in a sealed environment with a collision/reaction cell gas of choice such as helium or hydrogen. These atoms or molecules collide with any interfering polyatomic ions to reduce the ion energy to prevent them from entering the positively biased mass analyzer. The polyatomic interferents are larger in ionic radius than the monoatomic analyte, and are thus subjected to a greater rate of collision by the gas of choice, hence reducing their energy. Adjusting the RF voltage applied to all eight poles garners an effectively focused beam of ionized monoatomic analytes.

The mass analyzer used is a quadrupole type whereby applying varying RF and DC voltages across two of the four poles simultaneously, allows for the “tuning” of specific mass to charge ratios, which in turn results in element specific m/z detection by allowing those specific m/z ions to reach the electron multiplier at the end of the quadrupole. Because the range of applied voltages may be scanned very rapidly, a multitude of m/z ratios may be detected. The

4 quadrupole can therefore detect multiple elements virtually simultaneously when rapidly scanned. Elemental m/z ratios which are not selected are destabilized, never making it to the detector.

Size Exclusion Chromatography

SEC-HPLC was chosen in order to sort the CSF samples by molecular weight prior to elemental detection (the separation is effected by differences in hydrodynamic radii). A capillary HPLC approach, as carried out by Ellis [10] was initially tried for this study, however SEC-HPLC allowed for molecular weight distinctions “on-column,” rather than pre-treating the sample by means of 5 kDa molecular weight cut-off (MWCO) filtration. It was noted during CSF sample preparation that there was the slight possibility of losing 31P containing species within the desired molecular weight range (less than 5 kDa) by way of residual sample remaining within the 5 kDa MWCO filter element, so a SEC column with a MW range of 100 – 7000 Da was utilized. This way, sample is only filtered of large particulates which remained after sample preparation (Spin-X filter vials) and the column itself carries out the desired <5 kDa exclusion.

Molecules which are larger than the pore size of a size exclusion column’s packing material will elute quickly; those which are smaller than the pore size will move into the pores and particles, taking longer to elute from the column. Of course, caution must be employed because SEC separates based on the overall hydrodynamic volume of the molecule, taking into account its size and shape, more so than the sample’s molecular weight alone. In short, large molecules elute first, smaller ones elute last. The column end is connected via PEEK tubing to the

5 nebulizer of the ICPMS, resulting in an “online” and real-time analysis of eluted species via element specific detection.

Electrospray Mass Spectrometry

Because ICPMS employs a harsh ionization source, structural data regarding sample is completely lost, however as a compliment to the detection of 31P containing peptides and proteins, structural information is desired. As such, ESI-MS is then utilized in an “offline” manner: once SEC-HPLC coupled to ICPMS screens the sample and produces a chromatographic/ spectroscopic fingerprint of each sample, the samples are run on SEC-HPLC once more and then collected in the absence of the plasma. These collected fractions are then injected into the Agilent Technologies’ Chip Cube nano-LC, separated further, and then analyzed “online” with the ESI-MS, via an Ion Trap mass analyzer.

Sample is first loaded into the chip’s enrichment column by means of a capillary pump.

This “precolumn” serves to concentrate the sample prior to analysis. Upon fully loading, internal rotors through an on-chip six-way valve then switch the column flow to the analytical column which is then controlled by a nano pump, down and out from the built in electrospray needle tip. Figures 1a-c show this re-routing of solvent and sample flow on the microfluidic chip array.

Figure 1. Step by step diagram of microfluidic re-routing within the nanoLC Electrospray Chip. 1a- powered down, no solvent path is defined. 1b- during enrichment phase, capillary pump flow (p2/w) preconcentrates sample onto the enrichment column. 1c- during analytical phase, nano pump flow (p1) passes sample and solvent through the analytical column and out the built in electrospray needle tip for MS analysis [22].

Electrospray ionization is a soft ionizing technique, meaning that the overall structure of the sample analyte is left relatively intact, unlike being reduced to elemental form as is the case with ICP ionization. Ionized sample is then introduced into an electrified capillary which helps to focus the ion beam into the Ion Trap mass analyzer. The trap itself is essentially an

“electrode box” consisting of a ring electrode between two endcap electrodes. Holes on either end of the cap electrodes to allow ions to pass into and out of the “box.” An RF voltage (781 kHz) is then applied to the ring, keeping the end electrodes at ground. The RF potential difference oscillates in the confines of the ring and end electrodes, creating a quadrupole field.

As the potential is applied, ions at certain m/z ratios within the box are subjected to a

“potential well” and are trapped due to loss of energy from helium atoms acting as a collision

7 gas; increasing or decreasing the potential alters which m/z are trapped, effectively producing a full ion spectrum scan with high sensitivity [23]. Increasing the energy of analyte ions of interest by resonsance excitation within the trap causes fragmentation of the analyte by the ion-helium collisions already occurring. Application of an additional dipole across the endcap electrodes creates a destabilizing effect within the trap; ions caught in the “potential well” are destabilized and ejected from the trap in order of increasing m/z ratio [23]. Essentially, mass to charge ratios of analytical interest are selectively fragmented, destabilized, and those ions ejected from the trap to the mass detector, noting any fragmentation of parent and daughter ions within the trap which may ultimately strike the detector.

Experimental

18 MΩ cm-1 doubly deionized water was used to prepare aqueous Tris-Cl solutions and was purified using a NanoPure water treatment system (Barnstead, MA, USA).

Tris(hydroxymethyl)aminomethane was supplied by Acros Organics (New Jersey, USA); concentrated hydrochloric acid was purchased from Pharmco (Brookfield, CT, USA).

Acetonitrile and water used for chromatography were both high purity, HPLC grade reagents supplied by Honeywell Burdick&Jackson (Muskegon, MI, USA); Spin-X centrifuge tube filters (22

µm) were from Corning Inc. (Corning, NY, USA).

Control (healthy or normal), non-vasospastic, and vasospastic CSF samples used for this study were received from the University of Cincinnati Department of Neurology as lyophilized powders. Table (1) displays the sample name, sample disease type, as well as sample mass to volume conversion ratio. CSF was reconstituted into liquid form by first weighing 4 mL of

8 sample, utilizing provided mass to volume conversion values, followed by the addition of 400ul of Tris-Cl buffer (pH 7.35). Each sample was then filtered through 0.22µm centrifuge tube filters at 5000 RPM for 20 minutes. Samples were then loaded into sampling vials for SEC-HPLC and are injected without further molecular weight segregation.

Table 1. CSF sample nomenclature, disease type, and mass-volume conversion factor. Please note: Con, NV, and V represent the control, non-vasospastic, and vasospastic sample types respectively.

Sample Name Disease Type mg mL-1 109-Con Control (normal/healthy) 9.5 NV 71-01 Non-vasospastic 8.9 NV 111-1 Non-vasospastic 8.3 NV 113-1 Non-vasospastic 7.8 V 108-2 Vasospastic 6.1 V 109-2 Vasospastic 6.9 V 112-1 Vasospastic 5.7

Both ICPMS and ESI-MS experiments were carried out under standard analytical conditions.

The experimental detail for the ICPMS, ESI-MS and SEC-HPLC are given in Table 2.

Table 2. Instrumental parameters for the separation of 31P containing proteins in human CSF with ICPMS detection. Also described are the parameters for ESI-MS structural elucidation.

ICP-MS Conditions Instrument: Agilent Technologies 7500ce ICPMS (Agilent Technologies, Tokyo, Japan) Forward Power: 1500 W Collision/Rxn Cell Gas: He @ 3.5 mL/min QP Bias: -16 V OP Bias: -18 V

SEC HPLC Conditions Instrument: Agilent 1100 HPLC (Agilent Technologies, Palo Alta, CA, USA) Mobile Phase: 30 mM Tris-Cl, pH 7.35 Flow Rate: 0.5 mL/min Injection Volume 50 µL Column: Tricorn Superdex Peptide 10/300 GL (10 mm X 300 mm) (Amersham Pharmacia Biotech AB, Uppsala, Sweden)

ESI -MS Conditions Instrument: Agilent Technologies LC/MSD Trap XCT Ultra (Agilent Technologies, Germany) Drying Gas: Nitrogen (325°C) @ 6.0 L/min MS Capillary Voltage: 1980 V Skimmer: 40.0 V Capillary Exit: 100.0 V Trap Drive: 85.0 V MSn 2

Nano HPLC Conditions Instrument: 1200 Series HPLC-Chip/MS System (Agilent Technologies, Germany) Mobile Phase: A: 99.9% DDI H2O, 0.1 % FA B: 90.0% ACN, 9.9% DDI H2O, 0.1% FA Column: Agilent Zorbax 300 SB-C18 Enrichment Column: 4 mm x 75 µm x 5 µm Enrichment Flow Rate: 2.00 µL/min Analytical Column: 4 mm x 75 µm x 5 µm Analytical Flow Rate: 0.60 µL/min Gradient: 0.00-7.00 min: 90% B; 7.10-9.00 min: 3% B

Results

Chromatographic and Spectroscopic Data Analysis

The first step in the study is to screen the CSF samples for phosphorus containing peptides and or proteins. Figures (2), (3), and (4) showcase the chromatographic/spectroscopic profiles for each sample type.

Figure 2. 31P counts per second of “normal” control non-SAH CSF 109-Con sample. Trace obtained via SEC-HPLC with online ICPMS elemental detection. Please note the molecular weight regions.

Figure 3. 31P counts per second of non-vasospastic SAH CSF samples. Traces obtained via SEC- HPLC with online ICPMS elemental detection. Please note the molecular weight regions. 11

Figure 4. 31P counts per second of vasospastic SAH CSF samples. Traces obtained via SEC-HPLC with online ICPMS elemental detection. Please note the molecular weight regions.

31P containing species are shown in various molecular weight regions with visible similarities across each of the three sample types, most notably within the 1.4 kDa to less than than 0.20 kDa molecular weight range. Within each sample subset there are slight variations as shown by the overlaying the chromatograms, however the overall behavior of each sample initially appears to be consistent among the sample types.

This near uniform behavior is further investigated by means of the second step of this study: nano scale reversed phase HPLC coupled with ESI-MS. Sample fractions were collected offline from SEC-HPLC and analyzed on the coupled Chip Cube-Ion Trap set up with the parameters as given in Table (2). Figures (5) through (11) display the resulting ESI spectral data, specifically the overlay of base peak chromatograms (BPC) for a representative control, non- vasospastic, and vasospastic sample. A BPC showcases a higher signal to noise ratio compared to a normal total ion chromatogram (TIC) on the ion trap used for this study, and because there are fewer ions the chromatogram is less complex. Overlaying the BPC allows for the immediate

12 distinction of fingerprint identities across sample batches as well as sample types, despite having similar peak profiles and retention times from SEC-HPLC; co-eluting species are shown to be different entirely. Also it should be noted that in figures (5) through (11) are a sample of the results generated from Spectrum Mill (Agilent Technologies, Santa Clara, CA) database searching for those specific samples shown. Spectrum Mill’s Easy MS/MS Search option was used for this study. Search modification criteria included variable phosphorylation of serine (S), threonine (T), and tyrosine (Y). As this study was performed without enzymatic digestion, the

“no enzyme” digest option was selected. The protein database used for searching the species

“HOMO SAPIENS” was ipi.HUMAN [24]. Many hits were returned from the Spectrum Mill search, however only the first few entries are reported for the sake of brevity. The results shown were chosen from the highest scored, collectively. All additional Spectrum Mill findings are reported in Appendix 1.

Figure 5. (A) displays overlayed ESI BPCs of “normal” control non-SAH CSF (109-Con). Colors coordinate SEC-HPLC peaks with ESI spectra. (B) is the individual SEC-HPLC trace for 109-Con. Spectrum Mill hit report #1 is from peak 3; #2 and #3 are from peak 1

Figure 6. (A) displays overlayed ESI BPCs of non-vasospastic SAH CSF (NV 71-01). Colors coordinate SEC-HPLC peaks with ESI spectra. (B) is the individual SEC-HPLC trace for NV 71-01. Phosphorylated Spectrum Mill hit #2 is from ESI of peak set 2

Figure 7. (A) displays overlayed ESI BPCs of non-vasospastic SAH CSF (NV 111-1). Colors coordinate SEC-HPLC peaks with ESI spectra. (B) is the individual SEC-HPLC trace for NV 111-1. Spectrum Mill hit #3 is from peak 4; #4 is from peak 1, #5 is from peak 5; #6 is from peak 3

Figure 8. (A) displays overlayed ESI BPCs of non-vasospastic SAH CSF (NV 113-1). Colors coordinate SEC-HPLC peaks with ESI spectra. (B) is the individual SEC-HPLC trace for NV 113-1. Spectrum Mill hits #1, #2, and #3 are from peak 4; #4 is from peak 3

Figure 9. (A) displays overlayed ESI BPCs of vasospastic SAH CSF (V 108-2). Colors coordinate SEC-HPLC peaks with ESI spectra. (B) is the individual SEC-HPLC trace for V 108-2. Spectrum Mill hit #1 is from peak 2; #2 is from peak 1; #3 and #9 are both from peak 3

Figure 10. (A) displays overlayed ESI BPCs of vasospastic SAH CSF (V 109-2). Colors coordinate SEC-HPLC peaks with ESI spectra. (B) is the individual SEC-HPLC trace for V 109-2. Spectrum Mill hit #1 is from peak 2; #2 is from peak 5; #3 and #4 are both from peak 3

Figure 11. (A) displays overlayed ESI BPCs of vasospastic SAH CSF (V 112-1). Colors coordinate SEC-HPLC peaks with ESI spectra. (B) is the individual SEC-HPLC trace for V 112-1. Spectrum Mill hit #1 is from peak 7; #2 and #3 are both from peak 1; #4 is from peak 4.

Within each fraction peak from SEC-HPLC, despite having close similarities across the board, each sample type has markedly different phosphorylated proteins and peptides. The review fields generated by Spectrum Mill are as follows [25]: the numerical peptide score reports a numerical description of how valid the interpretation of submitted data is. For this study, most hits returned had peptide scores of 6-9 which describe a valid interpretation, providing other statistical indicators are high. The Fwd-Rev score is the difference between scores for top hits from a database search of the matched peptide sequence both in for forward sequential reading direction and the reverse direction Unfortunately because these values for figures (5) through (11) are relatively small, there exists the possibility of incorrect assignments. The SPI

(Scored Peak Intensity) % is the percentage of the MS/MS peak-detected spectral ion current.

For Ion Trap spectral data, an SPI % score above 70 is considered representative of good interpretations; the values for figures (5), (6), (7), (8), (9), (10), and (11) are all above 70. The

Spectral Intensity provides both a numerical and visual representation of how intense the protein’s concentration may be. Darker colors represent a higher concentration while the value is the average of the intensities for all peptides assigned to the protein. Sequence is the peptide amino acid sequence; modifications list the chosen variable modifications which returned as hits; retention time (RT) corresponds to the peptide’s retention time in the corresponding ESI spectrum for a given analyzed SEC-HPLC peak. MH+ Matched (Da) is the molecular weight of the matched peptide or protein; the accession number and protein name are used to further search for information regarding the identified species, utilizing a protein knowledgebase such as UniProt [26] or the International Protein Index [24]. The inclusive obtained results (see also Appendix 1) all appear to be in similar standing with the displayed

21 results in the above figures: moderate protein scores, very low forward and reversed scores, and very high SPI% and Spectral Intensity values and colors (dark red to yellow). As this study was performed in the vein of an “intact protein” analysis and database search, perhaps such low scores may be the result of not performing a digestion on the collected fractions prior to

Ion Trap analysis as the finer “cutting” of any phosphorylated proteins present may aide in generating peptide identification hits from the database Spectrum Mill searches. It should be noted that prior to adopting this described SEC-HPLC approach however, capillary-HPLC studies were performed and fractions of 5 kDa MWCO filtered CSF samples were tryptically digested and analyzed on the Chip-LC/MS system with no useful data generated. Another point of interest resides in the samples themselves. For this study, all CSF samples were freeze-dried rather than in a native liquid form as they were for previous capillary-HPLC work performed by

Ellis [10]. While lyophilized samples allow for a certain degree of control over working sample concentrations, this process might have caused some deterioration of very small proteins and or peptides. As these samples are lyophilized, and not the same samples used by Ellis et al., it is not surprising that the results of this study do not match hers. This also brings up an interesting point which proved to be a “logic obstacle” of sorts throughout the entire study: because this is a biological fluid, there remains the possibility that varying levels of phosphorus and phosphorylation could result from patient diet and environmental factors. This study appears to be relatively inconclusive in drawing a correlation between phosphorylated species in healthy patient CSF vs. diseased patient CSF and more work needs to be done with additional samples, both alone and pooled. Furthermore, patient history such as clinical, familial, and habitual, is necessary to potentially rule out uncontrollable variables within each sample set.

Protein Identification Discussion

Utilizing both the International Protein Index and UniProt, useful facts about some of the

Spectrum Mill generated IDs were gathered [24,26]. Discussed here are the findings for figures

(5) through (11). Because of relatively poor Fwd/Rev scores for the sequences identified, the overall protein IDs can only suggest their presence without full confirmation. Despite this, trends in protein function and nature seem to conform to sample disease type.

109-Con is the non SAH patient control CSF sample. As shown in Figure (5) Spectrum

Mill reported the presence of Importin-9 which functions as a nuclear transport receptor in nuclear proteins. Also reported in figure (5) are KIAA2018 and SIGLEC1, an isoform of

Sialoadhesin. The former ID is shown as a hypothetical protein, however UniProt names

KIAA2018 as "Basic helix-loop-helix domain containing protein KIAA2018." This protein appears to become phosphorylated upon DNA damage, which does not fit with the healthy CSF sample.

This does, however, imply that despite not having a SAH, even this "healthy patient" CSF sample may have come from someone possibly suffering other ailments. SIGLEC1 belongs to the sialic acid binding lg-like lectin family (SIGLEC) which mediate sialic acid dependent binding to various lymphocytes and may play a role in hemopoietic (production of blood cell components) functions. Overall, these identified proteins appear to be consistent and "expected" to be found in healthy patient non-SAH CSF.

Figure (6) shows Spectrum Mill results for the SAH non-vasospastic sample NV 71-01.

Only two hits were generated with #2 as the only one with phosphorylation as a PTM. Kalirin promotes the exchange of guanosine diphosphate (GDP) by guanosine triphosphate (GTP) and acts as a signaling mechanism inducer by activating certain members of the Rho GTPase family.

Kalirin has magnesium as its cofactor, of particular interest given one of the overall research goal of the Caruso Group is to delve deeper into the metal-binding characteristics of small metabolites by way of metallomics style approaches [27, 28, 29]. This may prove to be an interesting side path of research for the continuation of this project in the future. Also intriguing is that Isoform 2 of Kalirin is brain specific, being highly expressed in areas such as the cerebral cortex, amygdala, and hippocampus, however the isoform reported by Spectrum Mill,

Isoform 4, is expressed in skeletal muscle. This discrepancy could be described two different ways: as an error, it is likely realized in the very low Fwd/Rev score Kalirin received, as a forward and reverse sequence search may result in an erroneous identification or as a valid occurrence, consider the onset of SAH and the resulting traumatic disturbance to the blood- brain barrier. Any and all proteins, (phosphorylated or not) found in the bloodstream, despite their size, have the potential to travel through the blood vessels and end up in the now exposed subarachnoid space, and thereby detected.

Hit #1 in the database search of the SAH non-vasospastic sample NV 111-1, Figure (7), is a transcriptional activator known as GATA1, which is involved in the development of erythroids

(red blood cells). As the SAH non-vasospastic CSF samples come in contact with blood from

SAH, it is no surprise that a peptide of this nature is found. The structure of GATA1 contains a zinc finger [30] moiety, which again proves to be of metallomic interest. Also ANGPTL6 was reported, which is an angiopoietin-related growth factor which may play a part in the wound healing process and promote the chemotactic (migration towards or away from chemicals within an environment) activity of endothelial cells such as those lining blood vessel walls. This is not surprising because the lumen of a brain blood vessel ruptures upon SAH; the body then

24 secrets ANGPTL6 after such trauma in an attempt to repair the wound, and it is likely to appear within the diseased patient CSF. As expected, this protein does not show up in the normal CSF sample (109-Con).

Figure (8) displays findings for SAH NV 113-1. CSF1 Isoform, listed as Hit #1 continues the trend of blood-related molecular species appearing in the diseased patient CSF. This macrophage colony-stimulating factor plays a role in hematopoiesis of certain white blood cell populations. It also performs an immunological defense role within the body. The cancer/testis antigen CTAG2 and the RNA-binding motif RBMs2 both also are returned as being identified in this sample. Despite having a testicular tissue specificity, the former protein is also observed in 25-50% of head and neck cancers as well [26]. The latter ID appears to not have a prominent role in other functions beyond those of an RNA-binding motif. Spectrum Mill also reports Ubinuclein-1 as being in the sample, which acts as a regulator of senescence.

Both Titin and what UniProt alternatively names "Mediator complex subunit 14" are both noted in the SAH vasospastic sample V 108-2 (Figure (9)). Titin, cofactored with magnesium, is a key component in the assembly and functionality of vertebrate striated muscles, acting as a cross-linking bridge between the two halves of a sarcomere (the band present in striated muscle). It may also play a role in the interphase stage of cell reproduction.

UniProt describes various defects in Titin as playing a role in certain muscular afflictions, including muscular dystrophies, atrophies, as well as defects in cardiac muscle tissues. As mentioned for Kalirin found in SAH NV 71-01, even though Titin is not directly involved in neurological makeup, in the instance of SAH, proteins of many different types may find their way into the CSF. Not as impressive, mediator complex subunit 14 is a ubiquitous coactivator

25 involved in RNA polymerase II-dependent gene transcription [26]. Complement component C1q receptor is also suggested to be present and is involved in the enhancement of phagocytosis in macrophages and monocytes. More interestingly, it has also been linked to Alzheimer’s disease

[31], a very well studied neural degeneration, and is usually expressed in endothelial cells and platelets, once again both related to burst blood vessels on the brain surface after SAH.

The SAH vasospastic sample V 109-2 is displayed in Figure (10). Hit #1 does not register within UniProt's knowledgebase, and therefore no useful information could be found.

Furthermore, it is does not have phosphorylation as a PTM. Hit #2, Protein BAT4, also reports very little information despite having a phosphorylated PTM. Mitogen-activated protein kinase kinase kinase 9 (abbreviated as kinase x3 9), however, is described as being a magnesium cofactored activator of the JUN N-terminal pathway, which is a cellular response to environmental stress and proinflammatory cytokines [32]. Gene name MAP3K9, mitogen- activated proteine kinase kinase kinase 9 is expressed primarily in tumoric cells of the colon, breast, and esophagous. SLIT2 is reported as Hit #4 and for this study proves to be the most interesting hit described for sample V 109-2. Overall, SLIT2 is involved in the development and regulation of the CNS. SLIT2 plays a guiding role in cellular migration and is essential for neural development and acts as a neural signal modulator of sorts, preventing inappropriate signaling by axons from the olfactory bulb.

Figure (11) showcases FAT4 as the #1 Spectrum Mill Hit. Protocadherin Fat 4 is expressed in both the fetal and infant brain tissue and functions in the regulation of planar cell polarity as well as cellular adhesion. As a Cadherin, UniProt lists it as a “cell-cell interaction molecule,” and it is dependent on calcium for its hemophilic cell adhesion properties [33]. Also

26 described is HUWE1 482 kDa protein. UniProt provides information to various HUWE1 fragment proteins, however each fragment contains the E3 ubiquitin-protein ligase HUWE1 domain. This ligase mediates ubiquitination which controls certain protein functions and stability. This protein becomes phosphorylated upon DNA damage, and defects in HUWE1 can cause mental retardation in those afflicted. Stromelysin-2 is also identified. Also named

MMP10, Stromelysis-2 is cofactored with both zinc and calcium. Its function is described as the degradation of fibronecin and various other collagens. This occurs in certain physiological and disease processes such as embryo development and metastasis respectively [34]. Reported here in vasospastic patient SAH CSF, it may be that it plays a role in the breakdown of moieties now present in the diseased patient CSF.

Closing Remarks/Future Studies

Overall it has been shown that several phosphorylated proteins and peptides of less than 5 kDa are suggested to exist within normal, non-vasospastic, and vasospastic patient SAH CSF samples. The overall goal of this study is to discover relationships, if any, between protein IDs and disease types, in particular comparisons across the different sample types. This thesis reports only phase I of this study and as such, no definitive correlations may be drawn between disease types and phosphopeptide presence or absence. However, there does appear to be a distinct and logically rationalized trend in protein function and presence within normal, non- vasospastic, and vasospastic patient SAH CSF samples.

Further optimization of Spectrum Mill searching parameters are necessary to generate more favorable protein ID scores. In addition, other databases and search engines should also be investigated such as SwissProt and MASCOT respectively for additional protein hits. Also of interest is to explore phosphoproteins in CSF which are greater than 5 kDa. Current Caruso

Group studies by Jennifer Siverling strongly suggest the presence of large

(>5 kDa) selenoproteins in CSF by use of SEC-HPLC coupled to ICPMS [35]. A modification of the methods used for that study may prove beneficial to further phosphoprotein investigation. This present investigation also suggests further studies on CSF that may be worthwhile, namely in the field of metalloproteomics. A recent has already been initiated which focuses on total ion analyses of several biologically important metals (copper, calcium, magnesium, lead, and iron) within CSF [36].

Human Cerebrospinal Fluid is a complex biological matrix that has great potential to open new paths of neurological ailment and disease treatment discoveries. This study is one step to learning more about this biological fluid and the information it is likely to contain across healthy or diseased patient CSF. For this investigator, it has proven to be both a fascinating and intriguing sample to study and work with; the data gleaned by this investigation is very exciting.

Indeed, the future of this project and other CSF related projects within the Caruso Group will be a bright one.

References

1. Berg JM, et al, Biochemistry, 5th Ed. W.H. Freeman and Co. (2002) 2. Ubersax JA, Ferrell JE, Nature 8, 530, (2007) 3. Meseguer I, Molina JA, et al, J. Neural Transm 106 (1999) 4. Miyamoto Y, Yamamoto H, et al, Pediatrics International 46 (2004) 5. Lampl Y, Geva D, et al, J Neurol 245 (1998) 6. Sakamoto T, Takasu A, et al, The Journal of Trauma Injury, Infection, and Critical Care 58 (2005) 7. Redzic ZB, Preston JE, et al, Current Topics in Developmental Biology 71 (2005) 8. Gellein K, Roos PM, et al, Brain Research 1174 (2007) 9. Yuan X, Desiderio DM, J. Chromatogr. B 815 (2005) 10. Ellis J, Grimm R, Caruso JA, et al, J. Proteome Res. 7(11), (2008) 11. Wind, M.; Edler, M.; Jakubowski, N.; Linscheid, M.; Wesch, H.; Lehmann, W. D. Anal. Chem. 2001, 73, 2001 12. Wind, M.; Wesch, H.; Lehmann, W. D. Anal. Chem. 2001, 73, 3006-3020 13. Wind, M.; Kelm, O.; Nigg, E. A.; Lehmann, W. D. Proteomics 2002, 2, 1516- 1523 14. Wind, M.; Gosenca, D.; Kübler, D.; Lehmann, W. D. Analytical Biochemistry 2003, 317, 26-33 15. Pröfrock, D.; Leonhard, P.; Prange, A. Journal of Analytical Atomic Spectrometry 2003, 18, 708-713 16. Pröfrock, D.; Leonhard, P.; Ruck, W.; Prange, A. Analytical and Bioanalytical Chemistry 2005, 381, 194-204. 17. Krüger, R.; Kübler, D.; Pallissé, R.; Burkovski, A.; Lehmann, W. D. Anal. Chem. 2006, 78, 1987-1994 18. Krüger, R.; Wolschin, F.; Weckwerth, W.; Bettmer, J.; Lehmann, W. D. Biochemical and Biophysical Research Communications 2007, 355, 89-96 19. May TW, Wiedmeyer RH, Atomic Spectroscopy 19(5), (1998) 20. Shah M, Caruso JA, J. Sep. Sci. 28, p.1969, (2005) 21. Cadoux-Hudson, TAD, Pyne GJ, Clark JF, Emerging Theraputic Targets 3(3), (1999) 22. Agilent Technologies 23. Agilent 1100 Series LC/MSD Trap System Concepts Guide. Agilent Technologies 2005 24. http://www.ebi.ac.uk/IPI/IPIhuman.html 25. Agilent G2721AA Spectrum Mill MS Proteomics Workbench Application Guide. Agilent Technologies 2007 26. http://www.uniprot.org 27. Haraguchi, H.; Journal of Analytical Atomic Spectrometry 19: 5–14 2004

28. Szpunar, J.; The Analyst 130: 442–465 2005 29. Mounicou, S., Szpunar, J. and Lobinski, R.; Chemical Society Reviews 38: 1119–1138 2009 30. Visvader, JE; et al, Molecular and Cellular Biology 15,2, p. 634-641 1995 31. Webster, SD; et al, Experimental Neurology 161,1, p. 127-138 2000 32. Diener, K, et al; Proc. Natl. Acaf. Sci. USA 94, 9687-9692 1997 33. Goldberg, M; et al, J. Biol. Chem. 275 (32) 11, p. 24622-24629 2000 34. http://www.ncbi.nlm.nih.gov/sites/entrez 35. Jennifer M. Siverling, O. Dean Stuart, Joseph A. Caruso; “Selenium Speciation and Quantification in Cerebrospinal Fluid; An Insight to CSF Biomarker Development” (Pittcon 2009, Chicago, IL, Poster) 36. Jennifer M. Siverling, O. Dean Stuart, Leah Blackketer, Joseph A. Caruso; “Total Ion Analysis of Human Cerebrospinal Fluid for Biologically Important Elements, Utilizing Inductively Coupled Plasma Mass Spectrometry” (Pittcon 2009, Chicago, IL, Poster)

† http://www.holistic.ac.nz/free/boff03.html ‡ http://gundam.anime.net/

Appendix 1 – Additional Spectrum Mill results

Non-SAH CSF 109-Con Non-vasospastic SAH CSF NV 111-1

Non-vasospastic SAH CSF NV 113-1 Vasospastic SAH CSF V 108-2

Sample of vasospastic SAH CSF V 109-2 Sample of vasospastic SAH CSF V 112-1