The Pennsylvania State University

The Graduate School

Department of Chemistry

ANALYSIS OF BIOGENIC AMINE

NEUROTRANSMITTERS WITH CAPILLARY

ELECTROPHORESIS

A Thesis in

Chemistry

by

Justin R. Smith

© 2009 Justin R. Smith

Submitted in Partial Fulfillment of the Requirements

for the Degree of

Master of Science

December 2009

ii

The thesis of Justin R. Smith was reviewed and approved* by the following:

Andrew G. Ewing Professor of Chemistry Professor of Neural and Behavioral Sciences J. Lloyd Huck Chair in Natural Sciences Thesis Advisor

Tae-Hee Lee Assistant Professor of Chemistry and the Huck Institute of the Life Sciences

David Boehr Assistant Professor of Chemistry

Michael L. Heien Research Associate

Barbara J. Garrison Shapiro Professor of Chemistry Head of the Department of Chemistry

*Signatures are on file in the Graduate School

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ABSTRACT

The complex nature of brain function makes dissecting the roles of

neurotransmitters in physiological processes such as learning and memory difficult. The

use of a model system such as Drosophila melanogaster, the fruit fly, however, simplifies

the analysis of these roles with its less complex nervous system and the ease with which

it can be genetically manipulated to form mutants. This thesis describes the examination

of the biogenic amine neurotransmitters found within Drosophila head homogenates with

capillary electrophoresis (CE) coupled to both mass spectrometric (MS) and

electrochemical (EC) detection. Following an introduction to the fruit fly and the

analytical techniques utilized to study the samples, the development of a CE system with

MS detection is described in chapter 2. Once developed, the system was used to

investigate the efficacy of different sample preparation protocols for extracting biogenic

amines and other compounds of interest from the fly samples. Extraction protocols were

further studied in chapter 3 with CE coupled to EC detection. Additionally, the

interference of eye pigment and compounds from the cuticle of the fly was examined by

comparing wild type flies with both white mutant flies that lack eye pigment and a

dissected wild type brain. Lastly, the use of MS/MS detection is discussed to identify

other possible compounds of interest found within the fly head.

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TABLE OF CONTENTS

LIST OF FIGURES .......................................................................................................... v

LIST OF TABLES ............................................................................................................. vi

CHAPTER 1. Analysis of neurochemicals with capillary electrophoresis ....................... 1

Overview of neuronal communication ................................................................... 1 Drosophila as a model system................................................................................ 4 Capillary electrophoresis overview ........................................................................ 6 Detection ............................................................................................................... 10 Overview of the thesis .......................................................................................... 13 References ............................................................................................................. 15

CHAPTER 2. Sample preparation methods for the extraction of biogenic amines from Drosophila melanogaster analyzed using micellar electrokinetic capillary chromatography with electrochemical detection ................................... 16 Introduction ........................................................................................................... 16 Materials and methods .......................................................................................... 18 Results and discussion .......................................................................................... 21 Conclusions ........................................................................................................... 28 References ............................................................................................................. 29

CHAPTER 3. Capillary electrophoresis coupled to mass spectrometry to aid with

the identification of unidentified peaks ................................................................. 30

Introduction ........................................................................................................... 30 Materials and methods .......................................................................................... 31 Results and discussion .......................................................................................... 34 Conclusions ........................................................................................................... 44 References ............................................................................................................. 46

CHAPTER 4. Summary and Future Directions ............................................................... 46

LC MS/MS analysis of Drosophila samples ........................................................ 47 References ............................................................................................................. 51

Appendix. Experimental compounds of interest .............................................................. 52

v

LIST OF FIGURES

Figure 1-1: Schematic of action potential ........................................................................ 2 Figure 1-2: Typical CE setup ........................................................................................... 7

Figure 1-3: MALDI-ToF schematic ................................................................................. 12

Figure 2-1: Schematic of CE-UV setup ........................................................................... 20 Figure 2-2: Data from CE-MS experiments highlighting bradykinin elution. ................. 23 Figure 2-3: Fruit fly extraction using methanol and acidified methanol ......................... 25 Figure 2-4: Mass spectra highlighting two mass to charge ratios during different time

points ..................................................................................................................... 26

Figure 3-1: Electropherogram comparison for peak identification .................................. 36 Figure 3-2: Electropherograms of fly samples prepared using different extraction

protocols ................................................................................................................ 38

Figure 3-3: Effects of eye pigment presence on fly sample electropherograms .............. 41

Figure 3-4: Electropherograms of a white fly head and dissected WT brain ................... 43 Figure 4-1: Example of a tandem mass tag ...................................................................... 50

vi

LIST OF TABLES

Table 3-1: Elution times and areas of peaks present in acidified methanol and perchloric acid extraction of wild type flies as well as those for white fly homogenates extracted in acidified methanol ....................................................... 39

Table A-1: Table of biogenic amines and metabolites of interest, including their monoisotopic masses and structures ..................................................................... 52

Chapter 1

ANALYSIS OF NEUROCHEMICALS WITH CAPILLARY ELECTROPHORESIS 

The nervous system is made up of many neurons, which must communicate with

each other to regulate the various processes of the body. The release of chemicals known

as neurotransmitters is one manner in which these cells use to communicate.

Consequently, the ability to detect these neurotransmitters and their metabolites is thus

vital to understanding the mechanism behind neuronal dysfunction and disease.

Neurochemistry aims to provide an improved pharmacological, molecular, physiological

understanding of complex brain chemistries by probing the roles the transmitters play

with analytical techniques. Capillary electrophoresis (CE) is one such analytical

technique that enables the study of various chemical species ranging from amino acids

and peptides to natural products and drugs. Since the initial analysis and detection of

neurotransmitters with CE by Wallingford and Ewing1, the technique continues to push

the limits of detection necessary for neurochemical analysis. In recent years, CE remains

an applicable technique for investigating neuroscience and single cell neurobiology, able

to easily adapt to the changes in research focus.

Overview of Neuronal Communication

Neurons in the brain are connected to each other in complex networks, which

communicate via electrical and chemical signals. Stimulation of a neuron within one of

these networks elicits an electrical signal in the form of an action potential (Figure 1-1).

An action potential begins by a depolarization of the cell membrane from its negative

resting potential. In an unstimulated cell, membrane potential is dictated by the

1

Figure 1-1. Schematic of action potential transported down the axon of the neuron to the presynaptic terminal. Once the potential reaches the terminal, vesicles containing neurotransmitters fuse to the cell membrane, releasing their contents into the synapse. Neurotransmitters subsequently bind to receptors on a secondary neuron before being reuptaken by the original neuron. Reprinted with permission from reference 2. 2

2

concentration of ions inside and outside the cell. Ion channels maintain a high

concentration of potassium levels inside the cell, while sodium levels are kept low and

the negative potential of the membrane is imparted by the presence of chloride ions and

negatively charged amino acids and proteins.3 However, following stimulation, sodium

channels open and the ion rushes in, whereas potassium begins to leave the neuron. The

amount of sodium entering the cells is greater than the amount of potassium exiting,

causing the depolarization of the membrane to a positive potential. This electrical signal

is propagated down the axon (Figure 1-1-3) by the opening of sodium channels until it

reaches the presynaptic terminal. At the terminal, the electrical signal is converted to a

chemical one during exocytosis.

The process of exocytosis involves the release of compounds known as

neurotransmitters from vesicles into the space between two neurons (the synapse).

During exocytosis, vesicles in the presynaptic neuron move towards the plasma

membrane to fuse and release their contents. Transmitters are then able to bind to

receptors on the postsynaptic neuron once in the synapse. These receptors determine

whether the synapse acts in an excitatory or inhibitory manner.3 As their names imply,

the two interactions differ in the likelihood of an action potential being transmitted to the

next cell. Excitatory synapses increase the probability of the potential occurring within

the postsynaptic cell by causing sodium channels to open upon transmitter binding to the

receptor. In contrast, chloride channels often open at inhibitory synapses to lower

membrane potential further, making it harder for an action potential to occur.3 Following

receptor binding, the transmitters diffuse out of the synapse or are removed via reuptake

by the presynaptic neuron.

3

The transmitters released at these synapses can be a variety of molecules such as

amino acids, biogenic amines, and neuroactive peptides. Glutamate, glycine, and g-

aminobutyric acid (GABA) are the primary amino acid transmitters. Glutamate is often

an excitatory transmitter, but can also act in a modulatory capacity depending on the

situation.3 Glycine and GABA, however, are the major inhibitory transmitters, acting to

depress the propagation of action potentials following stimulation. Biogenic amine

transmitters include the catecholamines–dopamine, norepinephrine, epinephrine–as well

as serotonin and function in a diverse array of areas in the brain. Neuroactive peptides

are short amino acid sequences that have also been found to be pharmacologically active

in neurons. These molecules are capable of eliciting both excitation and inhibition,

depending on the targeted neurons. Because the interactions of these molecules with

neurons are complex in mammals, use of a model system such as Drosophila

melanogaster, which possesses analogous transmitters, is beneficial to examine their

roles with CE.

Drosophila as a Model System

Drosophila melanogaster, the fruit fly, is a well-studied model system that has

been used to examine complex biological processes conserved between the fly and

mammals.4-6 The use of flies is advantageous since they are small, easy to handle, and

inexpensive. In addition, they have a simple nervous system of ~200,000 neurons and a

sequenced genome with few functional redundancies. The reduced number of functional

redundancies means that if a gene is removed then there are likely no other genes that can

compensate for the loss of the initial gene, and thus the gene can be considered knocked

out. Because of these traits, genes relating to neurotransmission can be identified and

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knocked out with relative ease compared to similar methods in mice, which are more

genetically complex. Further, the flies have a short life cycle of 3–5 weeks, allowing the

mutations to be observed in the original genetically manipulated flies as well as in future

generations within a reasonable time frame. Phenotypes observed in mutated flies can be

more easily associated with functions that were lost or gained as a result of removing a

single gene. The ease with which genes corresponding to the creation and depletion of

biogenic amines can be altered also provides an advantage with the fruit fly. Further,

since the roles of biogenic amines are conserved between flies and mammals, traits,

which include learning and memory, that are regulated by these amines can be examined

by interrogating Drosophila.7, 8

The central complex of the Drosophila brain contains four major monoamines–

dopamine (DA), octopamine (OA), tyramine (TA), and serotonin (5-HT)–each of which

have been studied at length.7 These monoamines are believed to play roles in fly motor

control, emotion, associative olfactory learning, and conditioned courtship.8 Dopamine

and 5-HT are particularly fascinating because of the roles they play in learning and

memory9-11 and reward/addiction pathways12, 13. Because of their involvement in many

vital functions, the ability to regulate of these transmitters by metabolism is important.

Several mechanisms of neurotransmitter metabolism exist, oxidation via monoamine

oxidase (MAO), N-Acetylation by N-Acetyltransferase (NAT), and methylation via

catechol-o-methyltransferase (COMT), each of which deactivate functional amines such

that they can no longer interact with receptors. N-Acetylation appears to be the major

mode of deactivation in Drosophila.14, 15 Therefore, the metabolites created by NAT are

of particular interest to examine and quantify in the fly.

5

Both mutant flies as well as wild type Drosophila can be analyzed to gain a better

understanding of the mechanisms behind the responses of the flies to environmental

stresses as well as addiction to ethanol and other drugs of abuse. For example, the white

mutant, which lacks the red and brown eye pigment found in Canton-S flies, is more

quickly sedated upon exposure to ethanol. This mutant is also completely deficient in a

protein that functions as a tryptophan transporter16, while the partially rescued mini-white

mutant has the transporter present, but in lower quantities than that found in Canton-S

flies since fewer copies of the gene coding for the protein are present. Given that the

mutation interferes with the 5-HT metabolic pathway, it would be expected that levels of

5-HT and its metabolites would decrease for the white and mini-white varieties when

compared with the wild type.17

Capillary electrophoresis overview

Capillary electrophoresis (CE) is a technique ideally suited for analyzing

neurotransmitter content in Drosophila with its low sample volume requirements and fast

separation times.18 Since CE can be used to perform separations of samples ranging from

nanoliters to femtoliters of material, it is useful for the analysis of volume-limited

samples. Also, CE has large peak capacities, which allow for the separation of numerous

analytes simultaneously. Furthermore, its compatibility with sensitive detection

techniques allows for both mass and concentration-limited samples to be examined.

In a typical CE configuration (Figure 1-2A), a high voltage is applied to a

capillary such that the anode possesses a positive charge, while the cathode is negative.

6

Figure 1-2. A) Typical CE setup; a high voltage is applied to a capillary, separating analytes by electrophoretic mobility and EOF. B) The addition of surfactant to run buffer allows neutral molecules to be separated in the MEKC mode of CE. Analytes interact with the micelles and are further separated from similarly charged molecules. C) A 5-μm carbon-fiber microelectrode is held at an overpotential and inserted into the etched end of the capillary for electrochemical detection.

7

This difference in potential across the capillary allows sample molecules to separate

based on size and charge due to electrophoretic mobility. With electrophoretic mobility,

positively charged ions move towards the cathode. A secondary force, electroosmotic

flow (EOF), however, permits the movement of negatively charged ions and neutral

molecules as well as cations. EOF is generated within the capillary by the interaction of

the negatively charged silanol groups on its interior wall with cations present in the run

buffer to form a double layer of ions. The innermost layer consists of cations strongly

bound to the silanol groups of the capillary through an electrostatic interaction. This

layer remains bound to the capillary, while the cations in the outer layer remain solvated

and travel toward the cathode with the rest of the bulk solution. The cations are pulled

slightly ahead of the bulk solution due to the combined effects of EOF and

electrophoretic mobility, while anions are retarded in solution and have a lower mobility

than the bulk solution. Since neutral molecules are not affected by the applied electric

field, they travel with the bulk solution, arriving at the cathode after the cations but

before the anions.

The presence of the electric field in CE causes the flow of solution to be different

down the capillary than in pressure-driven systems such as liquid chromatography (LC).

EOF causes the flow of the bulk solution down the capillary to have a flat profile. In LC

systems, however, the hydrodynamic flow within the column is not uniform and friction

with the walls causes fluid velocity to be greatest in the center and near zero at the walls

resulting in a parabolic flow profile. This difference in flow causes CE to have minimal

analyte band broadening during the separation, allowing for a better resolution between

peaks. Another advantage of CE is the ability to alter conditions within the capillary to

8

change the separation and obtain better resolution and peak efficiencies. For example,

while decreasing capillary diameter lowers EOF,18, 19 it also reduces the effects of Joule

heating by increasing the surface area, helping to dissipate the heat generated by the

applied voltage. The reduction of Joule heating aids in achieving better resolution by

lowering the amount that analyte bands broaden during the separation. In addition,

smaller diameters allow for smaller injection volumes, decreasing the amount of sample

necessary for analysis. However, conventional CE has one disadvantage, its incapability

to differentiate between neutral molecules since they do not interact with the electric field

and travel at the same rate as EOF.

Micellar electrokinetic capillary chromatography (MEKC), a variation of CE,

allows for the neutral molecules to elute discretely from each other by introducing

surfactant to the run buffer. In CE, elution is based on the mobility, while elution in

MEKC is based both on the mobility of the analytes and their interaction with micelles.

As seen in Figure 1-2B, when a surfactant, such as sodium dodecyl sulfate (SDS) is

added to the run buffer, the elution order of the molecules is altered because of their

interaction with the formed micelles. In general, positive molecules interact with both

the negatively charged heads and hydrophobic tails of the micelles, causing them to elute

behind both the negative and neutral particles. Different neutral molecules interact

distinctively with the micelles based on differences in hydrophobicity, hydrogen bonding,

and charge.19 The negative molecules do not interact with the micelles and consequently

elute first despite the effects of EOF. By optimizing the pH and surfactant concentration

of the buffer, several analytes can be successfully separated and detected during the

course of an experiment.

9

Detection

There are a variety of detection methods that have been coupled to CE. Some

common detection methods associated with CE include laser-induced fluorescence (LIF),

electrochemical detection (EC), and mass spectrometry (MS), each of which provides

distinct information about the analytes being separated. LIF typically uses an argon-ion

laser to excite the fluorescent analytes in a sample with the intensity of light emitted a

function of concentration present in the sample. By absorbing energy from the laser, the

sample is excited to higher electronic energy states from the ground state at the excitation

wavelength of the sample. The sample then quickly emits the energy as fluorescence at a

longer wavelength within nanoseconds of the excitation. LIF is a sensitive technique that

is capable of detecting nanomolar concentrations. However, one disadvantage with

detector is that the sample usually needs to be natively fluorescent or tagged with a

fluorescent molecule in order to be detected.20

Electrochemical detection is another concentration-based detection technique

wherein a microelectrode is placed at the end of the capillary. Most often, carbon-fiber

working electrodes are used because of their resistance to biological fouling and large

working range of detectable analytes.21 In this scheme, only electroactive molecules are

detected as they elute, increasing the selectivity of the detection. Two modes of detection

are possible with electrochemistry, amperometry and voltammetry. With amperometry,

the electrode is held at constant overpotential to ensure the complete oxidation or

reduction of the sample in order to provide a measurable current. Voltammetric

detection, however, varies the potential at the working electrode. In a sample containing

multiple electroactive analytes with similar migration times, this scheme allows for an

10

additional means of identification since each analyte produces a characteristic

voltammogram.22

Though not as popular as LIF and electrochemistry, MS offers a unique advantage

when used as the detector following CE separation, the ability to identify the separated

species based on their mass to charge ratio (m/z). As shown in Figure 1-3A, matrix

assisted laser desorption ionization (MALDI) MS utilizes soft ionization, which is ideal

for analyzing the composition of biological samples because the sample is not

fragmented. As implied by the name, MALDI employs the deposition of a matrix

solution onto the sample, which has been placed onto a metal target. This matrix

crystallizes with the sample and improves the ionization process as well as reduces the

damage caused to the biomolecules by the nitrogen laser that is ionizing the spots on the

metal plate. When coupled to a time-of-flight (ToF) analyzer (Figure 1-3B), which uses

an electric field to accelerate ions and determine the time necessary to travel to the

detector, MALDI can determine a sample’s composition based on the m/z of its ions.

Electrospray ionization (ESI) is another soft ionization MS technique well suited

for proteomics and other macromolecules. This technique is often used to look at

biological macromolecules, without the need of a matrix to assist in ionization. During

ESI, the sample is first mixed with a solvent, and then introduced into a capillary vessel

with a fine tip (nozzle) at the end. When a high voltage is applied to the nozzle, the

sample molecules become charged and pass into an evaporation chamber to form a cone

(a Taylor cone) as they exit the nozzle. The cone, however, soon disperses as the

droplets containing these highly charged molecules repel and separate from each other in

11

A

B

Figure 1-3. A) In MALDI a laser beam, typically nitrogen, is used with an excitation of 337 nm to fire at the matrix and sample. Since the matrix absorbs the energy, the sample is kept intact as it is ejected to the detector. B) ToF MS is shown where ions are accelerated in a field of known strength which results in the same kinetic energy applied to all the particles. The time it takes for the molecules to travel a known distance is recorded. Since larger molecules travel slower, a separation occurs between different molecules and the m/z is determined. Reprinted with permission from reference 23.23

12

a fine spray since they all possess the same charge. The droplets subsequently undergo a

series of divisions because the solvent in the droplets gradually evaporates with the aid of

nitrogen gas introduced into the chamber. Because the charged molecules are forced

closer together, the droplet divides to minimize the repulsion between them. This process

continues until the droplet contains a single molecule, which then passes to the detector

where a mass spectrum is generated.

Overview of the thesis

CE is an ideal technique for examining the neurotransmitter content of the low

volume, concentration limited Drosophila head with its small injection volumes and

ability to be coupled to sensitive detectors. This thesis describes the analysis of

populations of homogenized fly heads with CE combined with both mass spectrometric

and electrochemical detection to gain insight into differences in transmitter levels

between wild type and mutant strains and attempt to identify previously unknown

compounds seen during the separations. The structures and monoisotopic masses for the

neurotransmitters of interest can be found in the Appendix.

The existence of unidentified compounds observed in the MEKC separation of

Drosophila heads necessitates the use of a secondary detection technique to determine

their identity. Chapter 2 details the use of CE coupled to MALDI-ToF MS detection.

Using MALDI as a detection source, it is possible to investigate several small molecules

and peptides found in the fruit fly. Samples of known standard compounds as well as fly

homogenates were separated on a CE system, and then spotted onto a MALDI plate for

detection at different time points. The coupling of CE with MS detection necessitated

that the homogenization protocol for the fly heads be altered since the normal extraction

13

medium, perchloric acid, suppresses analyte ionization during MALDI. The use of

acidified methanol was examined for extraction of neurotransmitters from Drosophila

head tissue and found to increase the number of unique peaks seen in the spectra over

extraction with methanol alone.

Since there was an increase in the number of peaks seen with the acidified

methanol during MALDI detection, the acidified methanol protocol was also employed to

prepare samples for MEKC coupled to electrochemical detection (MEKC-EC). The

comparison of these samples to those prepared with the original perchloric acid medium

is presented in chapter 3. Electropherograms generated for each preparation were

analyzed to determine which extraction medium yielded the best ratio of neurotransmitter

amount to number of transmitters able to be detected for Canton S wild type flies. White

mutants were also utilized in the study to determine how the presence of the eye pigment

in the wild type flies affected the separation profile. Acidified methanol was found to be

the better extraction method since more neurotransmitter peaks were observed in the

resulting electropherograms. Further, the lack of eye pigments in the white mutants was

found to have fewer peaks present in the separation with no overloading peaks present in

the profile. Finally, Chapter 4 will conclude this thesis with a summary of the

experiments presented herein as well as a discussion about the future directions of this

project.

14

References

1. R. A. Wallingford and A. G. Ewing, Anal Chem, 1989, 61, 98-100. 2. Anthropology.net, Neuron Synapse, Accessed June 22, 2009. 3. E. R. Kandel, J. H. Schwartz and T. M. Jessell, eds., Principles of Neural Science,

McGraw-Hill, New York, NY, 2000. 4. S. Doronkin, L. T. Reiter and P. M. Conn, in Progress in Nucleic Acid Research

and Molecular Biology, Academic Press, 2008, pp. 1-32. 5. N. Sanchez-Soriano, G. Tear, P. Whitington and A. Prokop, Neural Development,

2007, 2, 9. 6. D. S. Woodruff-Pak, J Alzheimers Dis, 2008, 15, 507-521. 7. M. Monastirioti, Microsc Res Tech, 1999, 45, 106-121. 8. W. S. Neckameyer, Learn Mem, 1998, 5, 157-165. 9. C. W. Harley, Neural Plast, 2004, 11, 191-204. 10. J. Kulisevsky, Drugs Aging, 2000, 16, 365-379. 11. M. Nomura, International Congress Series, 1995, 1088, 189-196. 12. S. Kobayashi and W. Schultz, J. Neurosci., 2008, 28, 7837-7846. 13. S. C. Tanaka, N. Schweighofer, S. Asahi, K. Shishida, Y. Okamoto, S. Yamawaki

and K. Doya, PLoS ONE, 2007, 2, e1333. 14. T. Roeder, Annu Rev Entomol, 2005, 50, 447-477. 15. R. J. Martin and R. G. Downer, J Chromatogr, 1989, 487, 287-293. 16. M. Nakamura, S. Ueno, A. Sano and H. Tanabe, Mol Psychiatry, 1999, 4, 155-

162. 17. J. Borycz, J. A. Borycz, A. Kubow, V. Lloyd and I. A. Meinertzhagen, J Exp Biol,

2008, 211, 3454-3466. 18. J. P. Landers, ed., Handbook of Capillary Electrophoresis and Associated

Microtechniques, CRC Press, Boca Raton, FL, 2007. 19. J. P. Landers, ed., Handbook of Capillary Electrophoresis, CRC Press, Boca

Raton, FL, 1996. 20. J. N. Stuart, N. G. Hatcher, X. Zhang, R. Gillette and J. V. Sweedler, The Analyst,

2005, 130, 147-151. 21. R. M. Wightman, Science, 1988, 240, 415-420. 22. F. D. Swanek, G. Chen and A. G. Ewing, Analytical Chemistry, 1996, 68, 3912-

3916. 23. N. H. M. F. Laboratory, MALDI,

http://www.magnet.fsu.edu/education/tutorials/tools/ionization_maldi.html, Accessed 06-01, 2009.

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Chapter 2

CAPILLARY ELECTROPHORESIS COUPLED TO MASS SPECTROMETRY TO AID WITH THE IDENTIFICATION OF

UNIDENTIFIED PEAKS

Introduction

Capillary electrophoresis (CE) is an efficient technique for separating samples

based on size and charge. Typically, both laser induced fluorescence (LIF) and

electrochemical detection are coupled to CE to identify compounds in the separation by

comparing them to known standards. One drawback to these detection schemes,

however, is that there are difficulties in determining the correct assignment of peak

identities in a complex sample when there are numerous unidentified analytes. Mass

spectrometry (MS) is ideally suited for this identification task because each analyte gives

a unique mass to charge ratio (m/z). Thus, when coupled to CE, MS data can be used to

help identify any unidentified peaks.

Matrix assisted laser desorption ionization time of flight (MALDI-ToF) is a form

of MS that can be used in conjunction with CE because it is a soft ionization technique,

which does not damage the analytes during ionization with a laser excitation source. This

phenomenon occurs due to the use of a matrix, typically either α-cyano-4-

hydroxycinnamic acid (CHCA) or 2,5-dihydroxybenzoic acid (DHB), which absorbs the

energy from the laser and distributes it such that the sample is ionized and allowed to

travel to the detector. The matrix also serves a secondary purpose, to preserve the sample

for hours, and in some cases even years, protecting it from oxidation1. This preservation

is particularly beneficial for sensitive biological samples prone to degradation or

16

oxidation and is not available when using other MS techniques. With MALDI-ToF, it is

possible to determine the m/z of important peaks in a sample. Once this m/z data has

been obtained for any peaks of interest, identification of the molecule that corresponds to

each peak can be obtained in a MS/MS mode of MALDI. With MS/MS, a molecular ion

of interest is bombarded with a heavy gas fragmenting the ion. These fragments can later

be combined to determine the structure of the whole molecule. The identity of these

compounds can then be used to quantitatively monitor changes in the amount of the

molecular species in samples with CE.

This chapter details the development of a CE device coupled to MS to help

determine the identity of unidentified peaks in samples of Drosophila melanogaster

heads observed in previous experiments using MEKC with electrochemical detection

(MEKC-EC). Drosophila is a model system in which the roles of biogenic amine

neurotransmitters in learning and memory and addiction are conserved between the fly

and mammals.2, 3 Because flies are inexpensive, possess a less complicated nervous

system, and are more easily genetically manipulated than mammals, they offer a more

simple means to examine how neurotransmitter and metabolite levels are altered upon

exposure to external stimuli or behavior changes. Once developed, the CE-MS system

was used to investigate the contents of populations of Drosophila heads. Both methanol

and acidified methanol extraction of fly heads were examined during this study to

determine which of the two yielded the most unique peaks in the mass spectrum.

Analysis of the data shows that the alteration of the extraction protocols used for

Drosophila caused differences in the number of peaks detected as well as the quantity of

compounds present in the samples under investigation.

17

Materials and Methods

Reagents. All compounds employed during experiments were purchased from Sigma (St.

Louis, MO) unless otherwise indicated and used as received. The N-Acetyl dopamine

(naDA) and N-Acetyl octopamine (naOA) were obtained from the NIMH Chemical

Synthesis and Drug Supply Program. All standards (see Appendix for compounds and

structures) were prepared as 10 mM stock solutions in 0.1 M perchloric acid and were

diluted to the desired concentration with additional perchloric acid solution.

Drosophila Strains and Homogenate Preparation. Canton S flies, a wild type

Drosophila strain, were maintained in the Han laboratory (Department of Biology,

Pennsylvania State University) and used to obtain head homogenates. Flies were

cultured on standard cornmeal/agar medium and collected between 2 and 4 days after

emerging from pupal cages. Prior to homogenization, populations of flies (40-50 flies

per population) were transferred to conical tubes and allowed to rest (approximately five

minutes) to minimize deviations from basal biogenic amine levels due to changes in

environment. The tubes were then submerged into liquid nitrogen to instantaneously

euthanize the flies and preserve the samples. Fly heads were dislodged from the bodies

by alternating the submersion into the liquid nitrogen and vortexing of the tubes as

needed until the flies were decapitated.

The fly heads were subsequently separated from the bodies and placed in a

commercial glass tissue homogenizer (Kontes Glass Co., Vineland, NJ). Heads were

homogenized until the tissue was broken up, and then homogenization liquid (either

methanol or acidified methanol–a mixture of 80% methanol/distilled water/acetic acid in

a 90/9/1 ratio) added. The sample was further homogenized and supernatant from the

18

mixture was collected and centrifuged at 4 oC through a 10-kDa filter at 14000 rpm for 90

minutes. The samples were subsequently speed vacuumed for pre-concentration

purposes before being reconstituted in 5 μL of 5% methanol.

Instrumentation and Analysis. The CE system with on-column UV-VIS detection

utilized in this study was built in-house as previously described4 and is shown in Figure

2-1. Briefly, 60 cm of fused-silica capillary (o.d. of 350 μm and i.d. of 75 μm, Polymicro

Technologies, Phoenix, AZ) was used for all separations. A window in the polyimide

coating was flame etched approximately 45 cm from the end of the capillary for on-line

UV-VIS detection. A second capillary and aluminum grounding wire were attached at

the detection end of the separation capillary with silver paint to introduce matrix solution

and to create a potential difference across the first capillary, respectively. Matrix solution

was introduced into the second capillary through a needle interface attached at the inlet of

the capillary from syringe loaded onto syringe pump (Harvard Apparatus, Holliston,

MA). This configuration permitted the continuous flow of matrix solution to the MALDI

spotter plate during the separation.

The separation capillary was filled with run buffer using a stainless steel reservoir

pressurized to 400 psi with helium. Separations were performed in 2.5 mM borate buffer

containing 30% acetonitrile, adjusted to pH 9.5. The buffer was filtered with a 0.2-μm

nylon filter (Alltech, Deerfield, IL) prior to use. An in-house LabView 7.1 (National

Instruments, Austin, TX) program was used to interface with the CE-UV system to

record separations and automate sample injections. Sample injections were performed

electrokinetically at 5 kV for 30 seconds. During the separation, the matrix solution was

19

Figure 2-1. A schematic of the CE-UV setup. The high voltage power supply is attached to an interlock box and operated by an in-house written Labview program. The supply applies a voltage across the separation capillary to separate analytes of interest. Simultaneously, a syringe pump is used to supply matrix through the second capillary. The separation capillary is run through the UV-VIS detector to verify that a separation is occurring. Both capillaries are joined together past the detector with silver epoxy, and a grounding wire (orange) added to create a potential difference across the capillary so a separation can occur. The magnified inset shows that analyte is spotted on a MALDI plate, which moved during the course of the experiment from spot to spot using an x, y, z stage.

20

set to flow at 0.5 μL/min from the syringe pump. Using an x, y, and z motorized stage

(SD instruments, Grants Pass, OR), the MALDI plate was moved from spot to spot every

five minutes during the separation collecting 1 μL per spot. Once the sample was

crystallized, the sample was taken to a MALDI-MS instrument for analysis.

MALDI-MS detection was completed on a M@LDI L/R (Waters, Milford, MA)

at the Proteomics and Mass Spectrometry Core Facility at the Pennsylvania State

University. Additionally, a Q-Star quadrupole MS/MS (Applied Biosystems, Foster City,

CA) in the Winograd lab (Department of Chemistry, Pennsylvania State University) was

also used for MALDI experiments. Mass lynx (Waters) and Analyst (Applied

Biosystems) software was used to examine the collected mass spectra from these

instruments.

Safety Considerations. An in-house safety interlock box was utilized to protect the user

from high voltage.

Results and Discussion

CE-MS System. An instrumental system was initially constructed to facilitate the

coupling of CE with MALDI-MS detection that incorporates the simultaneous delivery of

separated sample analytes and matrix solution onto a MALDI spotter plate. As shown in

Figure 2-1, the system consists of one capillary used to inject the sample and perform the

separation as well as a second capillary through which matrix solution is dispensed via a

syringe pump. The constant flow of solution from the matrix capillary functioned as the

cathode for the separation capillary completing the circuit for the voltage being applied.

A MALDI plate was then placed underneath the end of the two capillaries on a motorized

stage to allow the combined eluent and matrix to be spotted at regular intervals. Once the

21

CE system was constructed, separations in the capillary were verified using a UV-VIS

detector placed approximately 45 cm from the injection end of the capillary.

Validation of the CE System. Experiments were carried out to determine the matrix that

offered the best ionization of the fruit fly extracts, while minimizing the interference of

matrix peaks with analyte peaks. The most common matrix solutions, DHB and CHCA,

were tested to determine if any matrix peaks interfered with peaks of interest. Of the two,

the MALDI spectrum of CHCA alone was found to have the least amount of interference

with known compounds of interest. With CHCA chosen as the optimal matrix, a standard

sample containing several neurotransmitters and small peptides was prepared and

separated in the system to investigate whether the compounds could be detected. Figure

2-2 displays both unseparated and separated standard samples as detected by MALDI

ToF. Analysis of the unseparated sample produced a spectrum (Figure 2-2A) containing

a large number of distinguishable peaks indicative of the 100 μM standards (notably, leu-

enkephalin at m/z 556 and bradykinin at m/z 1060.8). Three standard compounds, leu-

enkephalin, bradykinin, and dopamine (m/z 154), were then monitored in the spectra

corresponding to 5-minute time intervals from the separation (plotted in a selected ion

electropherogram in Figure 2-2B). It was observed that of these compounds dopamine

eluted from the capillary first, spotted onto the MALDI plate between five and ten

minutes. Bradykinin exited the capillary between five and fifteen minutes after the

separation commenced and was therefore present in two different spots. This observation

is likely due to the compound beginning to elute at the end of one time interval and

continuing on to the next. The counts observed for bradykinin during these intervals

support this explanation. Changes in the sample spectra displayed in Figures 2-2C-F

22

Fi

gure

2-2

. Ana

lysi

s of

sta

ndar

d co

mpo

unds

and

pep

tides

with

MA

LDI-

ToF.

A) S

pect

rum

of s

tand

ards

and

pep

tides

whe

re a

rrow

s in

dica

te le

u-en

keph

alin

(m/z

556

) and

bra

dyki

nin

(m/z

106

0.8)

. B

) Sel

ecte

d io

n el

ectro

pher

ogra

m o

f bra

dyki

nin,

leu-

enke

phal

in a

nd

dopa

min

e (m

/z 1

54) e

lutin

g th

roug

h th

e ca

pilla

ry o

ver t

ime

and

dete

cted

usi

ng M

ALD

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-F) S

pect

ra o

f elu

ent s

pots

at t

ime

poin

ts 5

, 10

, 15,

and

20

min

utes

resp

ectiv

ely

whe

re a

rrow

s ind

icat

e th

e m

/z o

f bra

dyki

nin.

23

further illustrate the separation occurring in the capillary via the presence or absence of

bradykinin at each time interval.

Examining Extraction Method Efficacy. Following the validation of the CE-MS system

with standards, samples of fly head homogenates were separated and spotted onto the

MALDI plate for analysis. It was determined early on in the experiments that the

previously employed extraction protocol5, 6 was least optimal to use with the CE-MS

system. This protocol included the use of perchloric acid as an extraction medium, which

prevents the spotted eluent and matrix from crystallizing, thus precluding MALDI

detection. Consequently, new media had to be employed for the experiments to extract

biogenic amines and metabolites from within cells and vesicles in the fly heads. Two

types of extraction media, methanol and acidified methanol, were investigated to

determine which yielded the greatest number of peaks and therefore compounds from

fruit fly extracts (Figure 2-3). While both methods generated spectra with multiple

peaks, the fly sample extracted with acidified methanol had an increased number of peaks

present in the sample versus methanol alone, indicating that more compounds were

extracted. It is possible that methanol, as an organic solution, was capable of extracting

organic compounds present in the fly heads and solubilizing the lipid membranes of cells

and vesicles. Acidified methanol, however, was capable of extracting more compounds

since its methanol component could interact with organic compounds in the sample,

while the acid interacted with aqueous compounds to yield more peaks with both

extraction mechanisms.

24

Figure 2-3. Analysis of fruit fly heads homogenized using methanol (A) and acidified methanol (B) as extraction media, respectively. Samples were placed on a MALDI plate with CHCA matrix, then analyzed with MALDI-ToF.

25

Fi

gure

2-4

. M

ass

spec

tra o

f tw

o m

/z r

egio

ns d

urin

g di

ffer

ent

time

poin

ts f

rom

the

sep

arat

ion

of f

ruit

fly s

ampl

e ex

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ed w

ith

acid

ified

met

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l. S

pect

ra o

btai

ned

from

the

5-m

inut

e tim

e po

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f the

sep

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ion

deta

il th

e m

/z ra

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for 1

30 to

180

in A

and

690

to

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in B

. C

and

D d

ispl

ay th

e sp

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of t

he s

ame

two

regi

ons,

resp

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at t

he 1

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the

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t fly

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can

be

obse

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with

MS.

26

Upon identification of acidified methanol as the better extraction medium for the

fly samples, samples prepared in this manner were separated on the CE-MS system.

Figure 2-4 displays two different m/z ranges in the mass spectrum for five (A and B) and

ten-minute (C and D) time points in the separation. As was observed with the standard

separations, the spectra of the fly samples changed over time with the m/z 151.10 and

693.17 peaks prominent at five minutes, but absent in the ten-minute frame. In their

place, m/z peaks 140.56, 168.02, and 720.89 were dominant at ten minutes. The ability

to detect these peaks, though they do not correlate to current compounds of interest, is

significant because it illustrates the sensitivity of the MALDI detection, since the number

of moles present in the fruit fly samples is significantly lower than that present in

standards.

Preliminary MS/MS Analysis. In order to identify the peaks changing during the MALDI

detection of the fly extract separations, more experiments must be completed using

MS/MS instrumentation. Preliminary experiments with standard compounds using a Q-

Star MS/MS instrument were performed in collaboration with the Winograd Lab to

demonstrate the instrument’s ability to help identify the small molecules and peptides that

likely correspond to the unknown peaks observed with both MS and electrochemical

detection. The Q-Star employed a quadrapole mass filter to isolate a m/z window such

that only that a single analyte is analyzed at a time to help identify these compounds. In

this window, MS/MS was used to bombard the molecular ion into fragments that can

later be reassembled based on the fragmentation pattern to make a positive identification.

For instance, standards of dopamine and octopamine, which have the same molecular

weight and cannot be distinguished with MALDI-ToF alone, were identifiable as separate

27

species with this technique. Thus far, however, MS/MS studies of fly extract samples

were inconclusive because not enough sample was present to perform both the initial MS

data collection to select a peak of interest and MS/MS fragmentation.

Conclusion

A system was constructed that effectively couples the separation ability of CE

with the identification capabilities of MS. Both samples containing standard solutions

and fly extracts were examined using the CHCA matrix to investigate the identities of

previously unidentified peaks from MEKC-EC separations. In addition, acidified

methanol was determined to be the extraction medium, yielding the greatest number of

observable peaks following extraction of Drosophila head contents. Though preliminary

experiments were completed with a Q-Star MS/MS instrument, more experiments are

needed to confirm the identity of the previously unidentified peaks seen in MEKC-EC.

28

References

1. E. V. Romanova, S. S. Rubakhin and J. V. Sweedler, Analytical Chemistry, 2008, 80, 3379-3386.

2. M. Monastirioti, Microsc Res Tech, 1999, 45, 106-121. 3. W. S. Neckameyer, Learn Mem, 1998, 5, 157-165. 4. J. S. Page, S. S. Rubakhin and J. V. Sweedler, Analyst, 2000, 125, 555-562. 5. T. L. Paxon, P. R. Powell, H.-G. Lee, K.-A. Han and A. G. Ewing, Anal Chem,

2005, 77, 5349-5355. 6. P. R. Powell, T. L. Paxon, K. A. Han and A. G. Ewing, Anal Chem, 2005, 77,

6902-6908.

29

CHAPTER 3

EXAMINATION OF DIFFERENT EXTRACTION PROTOCOLS FOR DROSOPHILA MELANOGASTER HEADS USING MICELLAR ELECTROKINETIC CAPILLARY CHROMATOGRAPHY WITH

ELECTROCHEMICAL DETECTION

Introduction

The fruit fly, Drosophila melanogaster, has been widely used to study

neurobiology because of its small size, short life cycle, and less complex nervous system.

In addition, many aspects of sensory processing and higher order brain functions are

highly conserved between Drosophila and mammals making them an invaluable resource

for understanding neuronal processes and behavior. Furthermore, the roles of biogenic

amine neurotransmitters such as dopamine (DA) and serotonin (5-HT) are also conserved

between fruit flies and mammals in the central nervous system1 with respect to the

regulation and modulation of learning and memory as well as reward and addiction.2

In order to better understand the roles of these neurotransmitters, micellar

electrokinetic capillary chromatography with electrochemical detection (MEKC-EC) has

been used to distinguish between and monitor changes in biogenic amine levels in

Drosophila.3, 4 MEKC is a mode of capillary electrophoresis (CE) wherein the addition of

micelles to the run buffer enhances the selectivity between the similarly charged and

sized biogenic amines. Furthermore, selectivity in the analysis is also imparted by the

use of amperometry as the electrochemical detection scheme since non-electroactive

species are not detected. With this technique, the content of biogenic amines and their

metabolites extracted from the head tissue of different flies can be examined. The

medium employed for this extraction is particularly important for the analysis since it

30

dictates the types and amounts of compounds obtained during the homogenization of the

fly heads. For instance, because the perchloric acid is unable to interact with the lipid

membranes of cells and vesicles to free the biogenic amines from these compartments,

the mechanical action of the tissue homogenizer’s pestle to break up the cellular tissue is

the primary means to extract these aqueous compounds. The use of both acid and an

organic medium such as the acidified methanol utilized in chapter 2, however, allows for

the interaction of the acid with aqueous compounds, while also allowing the methanol to

interact with the lipids found in the cellular and vesicular membranes.

This chapter describes the investigation of two different extraction protocols to

determine which yielded the greatest amount of detectable neurotransmitters and

metabolites out of the fruit fly heads with MEKC-EC analysis. It was found that the

acidified methanol extraction of wild type flies permitted the detection of serotonin (5-

HT) with the borate/sodium dodecyl sulfate (SDS) run buffer, which had been absent

with previous extraction techniques.3 In addition, electropherograms generated from

homogenized populations of wild type flies were compared to those obtained from

populations of mutants with no eye pigment (white) to examine the hypothesis that the

eye pigment of the wild type fruit fly head causes interference with the detection of the

desired neurotransmitters. Further, separation profiles for the white mutants were also

compared to a wild type brain removed from the cuticle to further investigate how the

presence of cuticle compounds affects the electropherograms.

Materials and Methods

Reagents. All compounds were purchased from Sigma (St. Louis, MO) unless otherwise

indicated and used as received. The N-Acetyl dopamine (naDA) and N-Acetyl

31

octopamine (naOA) were obtained from the NIMH Chemical Synthesis and Drug Supply

Program. A 48% aqueous solution of hydrofluoric acid (HF) was obtained from Aldrich

(Milwaukee, WI). All standards (compounds and their structures are detailed in the

Appendix) were prepared as 10 mM stock solutions in 0.1 M perchloric acid and were

diluted to the desired concentration with additional perchloric acid solution.

Drosophila Strains and Homogenate Preparation. Canton S flies, a wild type

Drosophila strain, and white mutant flies were maintained in the Han laboratory

(Department of Biology, Pennsylvania State University) and used to obtain head

homogenates. These white mutants lack the red and brown eye pigments found in wild

type flies and were used to determine the effects of eye pigment on the separation profiles

observed with MEKC-EC. Flies were cultured on standard cornmeal/agar medium and

collected between 2 and 4 days after emerging from pupal cages. Homogenization

protocols were previously described in chapter 2. Briefly, populations of flies (40-50

flies per population) were transferred to conical tubes and allowed to rest (approximately

five minutes) prior to homogenization. The tubes were then submerged into liquid

nitrogen to instantaneously euthanize the flies and preserve the samples. A combination

of liquid nitrogen submersion and agitation by vortex of these tubes was subsequently

used to dislodge the fly heads from the bodies. The process was repeated until all flies

were decapitated.

The fly heads were subsequently separated from the bodies and placed in a

commercial glass tissue homogenizer (Kontes Glass Co., Vineland, NJ) Heads were

homogenized until the tissue was broken up, and then homogenization liquid (either 0.1

M perchloric acid or acidified methanol–a mixture of 80% methanol/distilled water/acetic

32

acid in a 90/9/1 ratio) was added. The sample was further homogenized to help extract

the biogenic amine content from the tissue. The supernatant was collected from the

mixture and centrifuged at 4 oC through a 10-kDa filter at 14000 rpm for 90 minutes.

The samples were subsequently speed vacuumed for pre-concentration purposes before

being reconstituted in 5 μL of 5% methanol.

Instrumentation and Analysis. The CE system with end-column amperometric detection

utilized in this study was built in-house and has been described previously5. Briefly, 45

cm of fused-silica capillary (o.d. 148 μm and i.d. 14 μm, Polymicro Technologies,

Phoenix, AZ) was used for all separations. Prior to separation, approximately 2 mm of

the polyimide coating was removed from the capillary tip to expose the fused silica,

which was placed in HF for 12 min while helium pressure was applied to the other end at

250 psi to enlarge the outlet. The same segment was then placed in a sodium bicarbonate

solution to neutralize the acid and then rinsed with distilled water. This enlargement of

the outlet capillary i.d. facilitated microelectrode placement and increased the

coulometric efficiency of the electrochemical detection.6

Capillaries were filled with separation buffer (25 mM Borate buffer, 50 mM SDS,

and 2% 1-propanol, adjusted to pH 9.5) using a stainless steel reservoir pressurized to

400 psi with helium. Buffer solutions were filtered with a 0.2-μm nylon filter (Alltech,

Deerfield, IL) prior to use. Injections were performed electrokinetically at 5 kV for 5 s.

In-column amperometric detection was carried out in a 2-electrode format with a 5-μm

carbon-fiber microelectrode held at +0.75 V versus a Ag/AgCl reference electrode

(World Precision Instruments, Sarasota, FL). Faradaic currents generated by the

oxidation of the analyte species in the samples were amplified with a current amplifier

33

(Keithley model 427, Cleveland, OH) and collected with a LabView 7.1 interface

(National Instruments, Austin, TX). Origin Pro 8 was used to plot the collected

electropherogram data to examine and identify peaks of interest. An in-house LabView

program was used to perform data analysis for the determination of individual peak areas

in all electropherograms.

Safety Considerations. An in-house safety interlock box was utilized to protect the user

from high voltage. Since HF could cause severe burns, it was used with extreme care.

HF was neutralized with sodium bicarbonate before disposal.

Results and Discussion

The small nature of the Drosophila brain and the compartmentalization of

neurotransmitters and metabolites within cells and vesicles render the initial tissue

extraction vital when analyzing these compounds from the volume-limited sample (brain

volume is approximately 5 nL). Traditionally,3-5 0.1 M perchloric acid was used as the

extraction medium during fly head homogenization due to its ability to minimize the

oxidation of the transmitters and metabolites of interest upon their exposure to oxygen in

the air and to inactivate the enzymes that degrade these compounds within the brain. The

acidic nature of this protocol, however, interfered with the MALDI detection scheme as

discussed in chapter 2. Fly samples were consequently homogenized with either

perchloric acid or acidified methanol. These samples were examined to determine

whether the large number of peaks observed with the acidified methanol preparation

during the MALDI experiments in chapter 2 translated to an increase in the number the

compounds perceived with MEKC-EC when compared to the perchloric acid protocol.

34

Protocol for Identifying Biogenic Amine and Metabolite Peaks. The identification of

biogenic amines and their metabolites in electropherograms generated from the

separation and detection of the fly head homogenates involved the comparison of

separations of a standard compound solution, the fly homogenate, and a spiked fly

homogenate. The standard solution contained the biogenic amines and metabolites of

interest at a concentration of 100 μM. This solution was separated prior to experimental

runs of fly homogenates with MEKC-EC to determine the migration times of each

compound for each new capillary. The elution order and relative migration times were

determined in advance for each compound individually. Fly homogenate samples were

then separated in the capillary, followed by a spiked fly sample. This spiked fly sample

consisted of the previously run fly homogenate combined with 1 μL of the standard

mixture. The inclusion of the standard mixture with the fly homogenate amplified the

peaks of interest present within the fly and aided in peak identification.

Once each of these samples was run, the collected data was plotted as

electropherograms, which were compared to each other to identify the peaks of interest in

the fly sample (Figure 3-1). While migration time comparisons could be made simply by

comparing the fly homogenate to the standard solution, the spiked sample accounted for

changes in the separation profile caused by the presence of additional unidentified peaks.

Using the standard solution and the spiked sample, the presence of biogenic amines such

as serotonin (5-HT, orange arrows) and dopamine (DA, black arrows) could be identified

in the fly as seen in Figure 3-1B.

35

Figure 3-1. Electropherogram comparison for peak identification in fly head homogenate samples. A) A separation of a standard mixture (blue trace) is compared to both a fly sample (red trace) and a spiked fly sample (green trace) to determine the identity of compounds of interest within the fly sample based on migration times and the presence of unidentified electroactive molecules within the sample. B) Magnification of the boxed in A highlighting the 5-HT (orange arrows) and DA (black arrows).

36

Comparison of Extraction Media. These identification procedures were used to compare

the extraction capabilities of perchloric acid and acidified methanol to determine the

optimal extraction solution. The electropherograms for the two protocols both displayed

multiple peaks generated by the oxidation of electroactive species at the microelectrode,

indicating that each medium was able to extract compounds from the fly heads (Figure 3-

2). As shown in Table 3-1, perchloric acid and acidified methanol solutions were both

effective in extracting DA, octopamine (OA), and the DA precursor, L-DOPA from the

sample. However, upon closer inspection of the acidified methanol sample separation,

this preparation method was found to yield a greater number of distinguishable peaks.

Though many of those peaks remain unidentified at this point in time, 5-HT was notably

detected with this extraction method, eluting at 280 s, while it was absent in the

separation of perchloric acid sample. The identity of this peak as serotonin was

confirmed by running standard solutions prior to each fly separation as well as a spiked

sample of the fly with the standards.

Another interesting difference between the sample preparation protocols involved

the size of the detected L-DOPA peak in the electropherograms. For the perchloric acid

preparation, the L-DOPA peak was noticeably larger than that observed with acidified

methanol. This phenomenon was not exclusive to L-DOPA, but was also apparent for the

other peaks detected with this extraction medium. These results could indicate that while

acidified methanol was capable of extracting more material from the fruit fly heads,

perchloric acid was more efficient for extraction of certain compounds such as L-DOPA.

37

Figure 3-2. Electropherograms of wild type flies extracted with acidified methanol (red trace) and perchloric acid (blue trace). The appearance of 5-HT is highlighted in the acidified methanol separation only, while L-DOPA is present in both traces.

38

Table 3-1. Elution times and peak areas present after sample preparation with perchloric acid solution or acidified methanol solution for wild type (WT) flies, as well as white flies extracted with acidified methanol (n=3 for all sample types). Resolved peaks of known compounds are listed below as well as various unidentified peaks for each separation. Separations following sample preparation with acidified methanol result in the greatest number of peaks, whereas sample preparation with perchloric acid solution results in more peaks having a greater amount of material present in them. Perchloric acid

WT Acidified methanol WT Acidified methanol white

Compound Time (s) Area (pC) Time (s) Area (pC) Time (s) Area (pC)Serotonin NA NA 280 9.85 276 3.53 Dopamine 380 31.1 380 2.22 NA NA Catechol 824 5.78 821 5.03 822 5.88 Octopamine 411 103 411 3.57 NA NA L-DOPA 1050 2,730 1057 91.0 1049 14.7 Unidentified Peak 1 321 4.92

2 342 5.40 345 3.60 3 444 53.1 447 1,250 447 2.35 4 573 92.0 573 23.1 571 19.2 5 597 77.0 6 600 7.97 610 48.4 611 27.9 7 1095 822 8 1263 2,350 1275 78.0 9 1347 3,850 685 9.95

39

The more efficient extraction of L-DOPA and other compounds by perchloric acid might

be caused be more rapid denaturing of the enzymes that cause the degradation of the

biogenic amines when compared to acidified methanol. Another possibility might be that

the larger peaks observed with the perchloric acid extraction were simply multiple

compounds that co-eluted, generating a larger single peak rather than several smaller

ones. This hypothesis might have merit as there was a second peak next to L-DOPA in

the acidified methanol separation that was not present in the perchloric acid extraction of

the wild type fly populations.

Influence of Eye Pigment on Separation Profiles. During the examination of the

separations for wild type fruit flies, numerous unidentified peaks were found to be

present. It has been postulated that at least some of these peaks were due to the eye

pigments found in the Canton S flies. To investigate how many of the unidentified peaks

observed in the electropherograms result from eye pigments, acidified methanol

extractions of both wild type and white mutant flies were carried out followed by MEKC-

EC analysis. Wild type flies are known to have two different eye pigment types, one red

and one brown. The red eye pigment comes from a pterin molecule called pteridine

(Figure 3-3A) that is soluble in organic solution. While the brown pigment comes from

ommochrome (Figure 3-3B), which is soluble in aqueous solution. The structures of

these molecules are electroactive and can be oxidized at the detection electrode. Further,

the molecules also have several amine sites that are capable of reacting with other

molecules as well. Finally, these molecules are likely present at higher concentrations

than the neurotransmitters being investigated. Thus, the presence of these molecules

could result in large peaks in the electropherograms and might consequently obscure

40

Figure 3-3. Effects of eye pigment presence on fly sample electropherograms. A) Structure of pteridine, the red eye pigment. B) Structure of ommochrome, the brown eye pigment. C) Electropherograms of a wild type fly homogenate (blue trace) and a white mutant homogenate (red trace). In both samples, 5-HT and L-DOPA are identifiable with the extraction and separation parameters.

41

peaks of interest. Mutant white flies, however, lack these pigments and can serve as a

negative control for these separations.

A comparison of electropherograms from wild type and white Drosophila samples

is shown in Figure 3-3. The large unidentified peaks observed in the wild type sample

were no longer evident in the white fly separation; rather, only smaller, less pronounced

peaks were observed. Despite the diminutive nature of these peaks, 5-HT, DA, OA, L-

DOPA could be identified in the electropherograms (Figure 3-3C and Table 3-1) based

on comparisons of the white fly sample separations with both standard mixture and

spiked white fly homogenates. The changes observed in the white fly samples versus the

wild type flies could be explained in two ways. Either the white fly potentially had more

differences in the genome than simply those effects observed with eye pigment and might

have a smaller amount of neurotransmitters present, or the peaks that were absent might

be generated by the oxidation of eye pigment and other compounds. The determination

of the factors causing the differences in the electropherograms between the two samples

could not be established with the current data.

Effects of Cuticle Compounds on Separation Profiles. A further investigation was

performed using a single brain from a wild type fly and white mutant fly homogenates.

By dissecting the brain out of the wild type fly, not only were the effects of the eye

pigments on the separation removed, but any interference caused by compounds in the

cuticle was also eliminated. Figure 3-4 shows a comparison of the white mutant sample

(blue trace) versus a dissected brain (red) from a Canton S fly. In both separations, the

electropherograms contained fewer large peaks when compared with the wild type whole

head. However, in the white mutant, the peaks were also considerably smaller when

42

Figure 3-4. Comparison of electropherograms generated for a dissected wild type brain (red trace) and white head homogenate sample (blue trace). Because the white sample does not have large, overloading peaks as compared to the wild type brain sample, the cuticle was determined not to interfere with identifying peaks of interest in the electropherograms.

43

contrasted against the wild type fly brain extract. This result might support the

hypothesis that there are lower neurotransmitter levels in the white mutants as was

recently reported by Borycz et al.7 Because both samples of the whole head white flies

(containing cuticle) and the dissected brain (no cuticle) did not produce the large,

overloading peaks observed with the wild type whole head homogenates, it was unlikely

that any compounds present from the cuticle interfere with the separation itself, although

its rigid nature might inhibit complete disruption of the brain tissue during

homogenization.

Conclusion

The effects of 0.1 M perchloric acid and acidified methanol on fly head extraction

were examined using wild type Drosophila heads and MEKC-EC analysis. Sample

preparation with acidified methanol was found to lead to an increase in the number of

distinguishable peaks in the separation. Notably, this leads to the detection of 5-HT in

these samples, which was previously absent with the perchloric acid sample preparation

was used for separations in borate buffer. Sample preparation with perchloric acid

solution, however, appears to extract a larger a quantity of some compounds.

Furthermore, a comparison of electropherograms of wild type and white mutant fly

samples was used to determine that the large, overloading peaks observed with wild type

head homogenates were likely due to eye pigment molecules since these same peaks are

missing in separations of the white fly samples that do not contain the pigments. Lastly,

the cuticle was not determined to have a negative contribution to the separation when the

analysis of white fly head homogenates and a dissected wild type brain was contrasted.

44

References

1. R. Fernández-Chacón and T. C. Südhof, Annual Review of Physiology, 1999, 61, 753-776.

2. J. Bergquist, A. Sciubisz, A. Kaczor and J. Silberring, J Neurosci Methods, 2002, 113, 1-13.

3. T. L. Paxon, P. R. Powell, H.-G. Lee, K.-A. Han and A. G. Ewing, Analytical Chemistry, 2005, 77, 5349-5355.

4. P. R. Powell, T. L. Paxon, K. A. Han and A. G. Ewing, Anal Chem, 2005, 77, 6902-6908.

5. P. J. Ream, S. W. Suljak, A. G. Ewing and K.-A. Han, Analytical Chemistry, 2003, 75, 3972-3978.

6. S. Sloss and A. G. Ewing, Analytical Chemistry, 2002, 65, 577-581. 7. J. Borycz, J. A. Borycz, A. Kubow, V. Lloyd and I. A. Meinertzhagen, J Exp Biol,

2008, 211, 3454-3466.

45

CHAPTER 4

FUTURE DIRECTIONS

The preceding chapters of this thesis have focused on the coupling of capillary

electrophoresis (CE) with mass spectrometric and electrochemical detection to gain a

better insight into the roles of biogenic amine neurotransmitters in Drosophila

melanogaster. Because several roles of biogenic amines are conserved between

Drosophila and mammals, the ability to monitor changes in neurotransmitter levels is of

particular importance. In chapter 2, MALDI mass spectrometry (MS) was used as a

detection scheme to aid in the identification of unidentified peaks seen with MEKC-EC

as well as to investigate the effects of different extraction solutions on analyte content

obtained from fruit fly homogenates. Of the extraction solutions, acidified methanol was

found to give the most unique peaks from the fly samples that did not interfere with peaks

from the matrix or buffer solution. Since the use of acidified methanol solution was

found to increase the number of peaks observed from fly samples, the effects of this

extraction technique on samples analyzed with MEKC-EC were compared with samples

prepared using extraction with perchloric acid solution in chapter 3. While both

perchloric acid and acidified methanol solutions can be used to extract multiple

compounds present within Drosophila, a peak for serotonin was observed in the

separation following sample preparation with acidified methanol. Serotonin was not

detected with the current separation parameters using extraction with perchloric acid

solution. Further, compounds in the heads of white flies and dissected wild type fly

brains were also separated. The results of these experiments appear to indicate that the

46

outer cuticle of the fruit fly head as well as the eye pigment might play a role in

obscuring the separation of the desired neurotransmitters.

The use of electrochemical as well as mass spectrometric detection can be

combined in order to gain a greater insight into changes in physiological processes

dictated by biogenic amines. Since it has been shown that there are differences between

fruit fly heads and their extracted brains, it might be possible to apply MALDI MS to the

analysis of both fly head and brain samples and then compare the two spectra in order to

identify what compounds are present in the head, but not from the brain itself. However,

it might be difficult to obtain enough dissected brains for analysis to have a sufficient

number of moles of sample present on the MALDI plate to be detected. Typically for fly

population studies, 40-50 flies are used per preparation. Since the dissection of this many

fly brains by even the most adept person can take several hours, it might not be feasible

to perform such a study. If this brain preparation can be accomplished, however, it could

be used to demonstrate that there is value in using only Drosophila wild type brains for

analysis instead of whole heads. Another alternative to determine which compounds do

not correlate to the brain would be to use white mutants. The use of these mutants in a

sample generates a less complicated separation, allowing for a better focus on the

molecules of interest.

LC MS/MS Analysis of Drosophila Samples

Identification of more analytes of interest within Drosophila could be aided by the

use of liquid chromatography (LC) coupled to MS/MS on fruit fly extracts. LC MS/MS

allows direct coupling of the separation and detection steps in sample analysis without

loss of any material during transfer, which typically results in more sensitive detection.

47

Further, this technique is capable of identifying small molecules as well as proteins and

peptides present in the fly simultaneously. Once the identity of these molecules is

known, it may be possible to track changes in their levels between wild type flies and

mutants or upon exposure to different stresses such as drugs and alcohol.

While attempts have been made to perform LC MS/MS using Canton-S flies with

a QTOF Premier (Waters, Milford, MA), the high concentration of eye pigment

overloaded the column and did not yield any results. White flies were tested with this

technique as well since they lack eye pigments. These spectra, however, also proved to

be too complex for the software to identify small molecules and peptides. The inability

to resolve the spectra is due to the instrument’s use of a format in which five second time

frames are pooled and ionized. Consequently, LC MS/MS is more beneficial if the

separation does not contain numerous analytes that elute in the same time period.

However, since the entire fly head was used, there were numerous molecules within each

time point, making an accurate determination of their identity impossible.

To circumvent this result, a tryptic digest of the fruit fly homogenates prior to

analysis might aid in producing a simpler separation and a more accurate identification of

peptides and proteins of interest. Trypsin is an enzyme that digests protein by cleaving

peptides on the carboxyl group side of lysine or arginine residues. The resulting sample

following the digest leaves peptide fragments that can be analyzed and sequences can be

extrapolated to determine the identity of the proteins. If this were to be done, then the

preparation protocol would have to be altered by removing the filtration step such that the

proteins will remain intact for the digest to work. Otherwise the peptides could

potentially be broken up into fragments too small for accurate determination. This

48

process, however, will not affect the detection of the desired small molecules since they

do not have the cleaving sites the trypsin enzyme acts upon.

Once peptides of interest are identified, it may be possible to use a tandem mass

tag to attach to the molecules of interest. Depending on the structure of the compound of

interest, it will be possible to choose an appropriate tag such that when LC MS/MS is

performed it would be possible to see the fragment ion of the tag and its abundance

present in order to quantify the amount of peptide present in the sample. Tandem mass

tags permit simultaneous determination of the identity and relative abundance using

isobaric tags.1 These tags are attached to the amino terminus of lysine residues (see

example in Figure 4-1). Each tag consists of three parts, a mass reporter, a mass

normalizer, and a protein reactive group. The mass reporter section has a site built in to

the molecule for preferential cleaving using MS/MS, whereas the mass normalizer

ensures that each tag in a set has the same mass. Finally, a protein reactive group is

present in order to ensure the tag binds to specific sites on the peptide or protein of

interest. By tagging different analytes in this manner, it would be possible to compare

samples across different treatment types simultaneously in a single experiment or to

ensure replicates of one sample are the same.

49

Figure 4-1. An example of a tandem mass tag that consists of three parts: a reporter segment that fragments off with MS/MS, a normalizer to ensure isobaric tags, and a reactive group to attach to the peptide or protein of interest. Figure reproduced with permission from reference 2.2

50

References 1. A. Thompson, J. Schafer, K. Kuhn, S. Kienle, J. Schwarz, G. Schmidt, T.

Neumann and C. Hamon, Anal Chem, 2003, 75, 1895-1904. 2. Thermo Scientific, TMT Figures,

http://www.piercenet.com/Objects/View.cfm?type=Page&ID=B60171CD-5056-8A76-4E36-958F5C186495#tmtstruct, Accessed June 27, 2009.

51

APPENDIX

EXPERIMENTAL COMPOUNDS OF INTEREST

Table A-1. The following table has a list of neurotransmitters, structures, as well as their monoisotopic masses plus hydrogen (M+H).

Compound [M+H] (Da) Structure

Dopamine (DA) 154.08

Epinephrine (E) 184.10

Octopamine (OA) 154.09

Serotonin (5-HT) 177.10

Norepinephrine (NE) 170.08

Tyramine (TA) 138.09

52

Compound [M+H] (Da) Structure

N-Acetyldopamine (na DA) 196.10

3-Methoxytyramine (3-MT) 168.10

N-Acetylserotonin (na 5-HT) 219.11

N-Acetyloctopamine (na OA) 196.10

3,4-Dihydroxyphenylacetic acid (DOPAC) 169.05

Homovanillic acid (HVA) 183.07

5-Hydroxyindoleacetic acid (5-HIAA) 192.07

53

Compound [M+H] (Da) Structure

Catechol (Cat) 111.04

54