Analysis of Reproduction Proteins from - DiVA portal450437/FULLTEXT01.pdf · 1.2 Capillary...

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Analysis of Reproduction Proteins from Butterflies

Jiawei Xu

Diploma thesis

Division of Analytical Chemistry

Department of Chemistry

School of Chemical Science and Engineering

KTH Royal Institute of Technology

Stockholm, Sweden 2011

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Abstract

Male butterflies have some certain pathways to prevent the female butterfly which has mated with them from mating with other butterflies. Research shows that some proteins from the male butterflies may play an important role in this mechanism. To investigate how the pathway works, the proteins contained in the spermatophore which is injected into the female butterfly by the male one during mating are very important.

In this study, the butterfly spermatophore proteins were mainly studied. The proper procedures to obtain the spermatophore from the female body were developed. Since capillary electrophoresis (CE) has many advantages for separation and detection of low amounts of sample, it was used in this study in order to separate the proteins obtained from spermatophores. Finally, Mass Spectrometry (MS) was performed to analyze the proteins after separation in order to identify the proteins.

This thesis mainly introduced the following points:

1. The method for obtaining spermatophores from butterfly bodies and the procedures for extracting proteins from spermatophores.

2. Optimized methods and conditions of Capillary Zone Electrophoresis (CZE) for butterfly spermatophore proteins. Several CZE methods and sets of conditions were compared in order to find the optimized ones.

3. Protein samples were applied to Mass Spectrometry (MS) to try to analyze and identify them. Moreover, the effect of using ZiptipsTM for sample pretreatment was discussed.

Keywords: Butterfly, reproduction proteins, capillary electrophoresis, mass spectrometry.

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Contents

1 Introduction 5

1.1 Background 5

1.2 Capillary Electrophoresis 5

1.2.1 Basic principles of Capillary Electrophoresis 5

1.2.2 The modes of Capillary Electrophoresis 7

1.3 Fluorosurfactant 9

1.4 Mass Spectrometry 10

2 Materials and Methods 12

2.1 Materials 12

2.1.1 Samples and reagents 12

2.1.2 Equipments and instruments 12

2.2 Methods 13

2.2.1 Obtaining spermatophore from butterfly 13

2.2.2 Extraction of proteins from butterfly spermatophore 14

2.2.3 Preparation of standard protein samples 15

2.2.4 Capillary Zone Electrophoresis 15

2.2.5 Mass Spectrometry 16

3 Results and Discussion 17

3.1 Capillary Zone Electrophoresis using a buffer without fluorosurfactant 17

3.1.1 HP commercial instrument 17

3.1.2 Homemade instrument 21

3.2 Capillary Zone Electrophoresis using a buffer with fluorosurfactant 23

3.3 Mass Spectrometry 25

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4 Conclusions 30

4.1 Optimizing the conditions of Capillary Electrophoresis 30

4.2 Conclusion from Mass Spectrometry result 30

4.3 Further research 31

5 List of abbreviations 33

6 Acknowledgements 34

7 References 35

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1 Introduction

1.1 Backgrounds

There are always conflicts between male and female animals. One of these conflicts of importance for insects is the mating frequency. Male insects always desire that the females only mate with him, because in this case his genetic information will be passed down to his offspring without any risk. However, on the other hand, the female insects want to mate with as many males as possible, since the spermatophores which are injected into their bodies by the males during mating contains many nutrients and some other materials which the females can benefit from.(1) Under this situation, male insects have to find some ways to prevent the females from mating with other males. There are different kinds of solutions for the males. One method is to guard the female after mating and drive away other males. The other one is to transfer a plug before finishing the mating, which is just a kind of physical obstacle and it will physically intercept other males’ sperm. It is discovered recently that when transferring the spermatophore, some proteins might be transferred as well at the same time.(2,3) These proteins would be responsible for a new strategy of preventing the female from mating with other males. As a result, separating and analyzing the reproductive proteins in the spermatophore would provide some important clues for the study of insects’ habits and physiology.(4)

This study was a cooperation project with Johanna Liljestrand Rönn at Zoology, Stockholm University(SU) and all butterfly samples used in this project were acquired from Christer Wiklund, SU.

1.2 Capillary Electrophoresis (CE)

The separation technique used in this study was capillary electrophoresis (CE). The technique of CE was developed in 1980. Since then, the principles and techniques of CE have been greatly developed. As we know, CE has many advantages such as low injection volume, fast separation and high sensitivity.(5) As a result, CE has become a widely used separation method in chemical, biological and medical science. The separations of analytes from small molecules to living microorganisms, including bacteria and virus, have been reported as successful.(6,7)

The advantages of CE are:

1. The inner diameter of capillary is small and the current for electrophoresis is low;

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2. Since the distance from the center of capillary to the capillary wall is short, the heat generated during the electrophoresis can be dispersed quickly. This will prevent the electropherotic bands from spreading;

3. CE has high resolution which is the advantage of electrophoresis, and the analysis time required is short, which is the advantage of high performance liquid chromatography (HPLC);

4. The amount of reagents consumed during CE is low and meanwhile the detection sensitivity is high. Furthermore, biological samples are less easily destroyed during CE;

5. CE is easy to use and it has high degree of automation.

1.2.1 Basic principles of CE

A CE instrument always contains a high voltage supply, a capillary, a detector and two vials which can contain the electrodes of the high voltage supply and the ends of the capillary (Fig 1).

Fig 1. The model of a CE machine

The inner wall of a capillary contains silicon hydroxyl groups, which can dissociate into –SiO‐ after the capillary is flushed with sodium hydroxide. The –SiO‐ groups would affect the solution near the wall to form a double electrical layer. In an electric field, all charged ions will move towards oppositely charged electrode. This effect is called electrophoresis. Meanwhile, the positively charged buffer layer formed due to the negatively charged capillary wall will move towards the negatively charged electrode at the end of the capillary and it will force the whole buffer to migrate together. This effect is called Electroosmotic Flow (EoF) (Fig 2).

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Fig 2. The generation of EoF

As a result, the moving speed of all charged ions in the capillary is determined by both their electrophoresis velocity and EoF. The directions of electrophoresis for positively charged ions and EoF are the same; the directions of electrophoresis for negatively charged ions and EoF are opposite and the neutral ions do not have electrophoretic migration. This causes the ions in the capillary to be separated. The positively charged ions come to the outlet first; neutral analytes come later while negatively charged ions come latest (Fig 3).

Fig 3. Running model of CE

1.2.2 The modes of CE

Capillary electrophoresis is an open platform and can be combined with many other analytical systems. Thus, the analytes for capillary electrophoresis might be affected by several different kinds of forces during the electrophoresis process after the combination

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and modification with other methods. According to the forces that analytes in the solution are influenced by, CE has several modes:

1. Capillary Zone Electrophoresis (CZE)

Since the weights and the charges that molecules have are different, under the same voltage, different kinds of molecules have different moving speed, and they can be separated while the electrophoresis time passes by. This is the principle of CZE. CZE is the most widely used mode of CE. It is mainly used for the separation of proteins, peptides, nucleotides and some other kinds of analytes which carry charges in the buffer solution.(8,9)

2. Micellar electrokinetic chromatography (MECC)

Adding surfactants to the background electrolyte, which can decrease interfacial tension between two liquids, and when the concentration of surfactant is above the critical micelle concentration, the surfactants will gather under the hydrophobic interaction and form a micellar phase. The migration velocity of analytes in the solution phase and in the micellar phase are different, moreover, the distribution coefficiency of analytes between these two phases are different during electrophoresis process. As a result, analytes get separated under both the effects of electrophoresis and the distribution to the micelles. MECC is mainly used for the separation of neutral molecules and small molecule pharmaceuticals.

3. Capillary gel electrophoresis (CGE)

Inserting a gel for gel electrophoresis into the capillary will make the molecules moving within the gel medium. Besides the separation principle of CZE that molecules are separated according to their charge‐mass ratios, the gel medium also has the effect of a molecular sieve. CGE is used for the separation of proteins, which have different molecular sizes, and some low molecular weight biological molecules such as oligonucleotides, RNA and DNA fragments.

4. Capillary iso electrical focusing (CIEF)

For CIEF, ampholytes are added into the electrophoresis buffer in order to form a pH gradient. When the protein is in the buffer where the pH is lower than its pI value, the protein will be positively charged and if the pH is higher than its pI, the protein will be negatively charged. So under the electrical forces and the pH gradient, the protein would gather to the point where the pH is equal to its pI value at the end of the electrophoresis process. This mode is only available for the peptides and proteins which have pI values.(10)

5. Capillary isotachophoresis (CITP)

For CITP, leading and trailing electrolytes need to be used as the background electrolytes. This method depends on the difference of mobilities of different molecules and is mainly used for sample concentrating.

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6. Capillary electrochromatography (CEC)

CEC is the combination of capillary electrophoresis and high performance liquid chromatography (HPLC). By introducing a stationary phase like the one used in HPLC into CE, the analytes in the sample will interact with the solid phase, and they can get separated according to the distribution between the mobile phase and the stationary phase.

Besides these modes, there are some other modes such as non‐aqueous capillary electrophoresis and affinity capillary electrophoresis. They have been rapidly developed during recent years as well.

1.3 Fluorosurfactants

When using CE for protein separation, there is one huge problem. That is, that the adsorption of proteins to the capillary inner wall is difficult to eliminate. Several methods have been developed to get rid of the adsorption, such as adding certain salts to the buffer, extreme buffer pH, capillary wall coatings and the use of some buffer additives. In this study, first extreme pH values of the buffer were applied. Furthermore, for parts of the examination of CZE conditions, a fluorosurfactant was added into the buffer, which is treated as buffer additive here.

A fluorosurfactant is a kind of surface active agent, like the dish cleaner we use at home. It has a hydrophilic head and a hydrophobic tail, or vice versa. The fluorosurfactant used in this research was one with a hydrophilic head, which was positively charged in the buffer. When it is added into the buffer, it will form a double layer automatically under the hydrophobic interaction between the tails near negatively charged capillary wall. Thus, the buffer around the double layer of fluorosurfactant will be negatively charged due to the electric attraction effect. So if a reversed voltage is applied, which means the anode is placed at the outlet side, an EoF will also be generated (Fig 4).

Fig 4. The principle of CE after adding fluorosurfactant

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High pH buffer can boost the EoF, and proteins with low pI values will be negatively charged and the highly negatively charged capillary wall will repel them. However, the proteins with high pI values will thus be positively charged and can be attracted to the negatively charged wall. Moreover, there are always some regions on the proteins that are hydrophobic. Under the effect of all these factors, proteins are easily gathered and adsorbed onto the capillary wall.

The main reason for why fluorosurfactant can decrease the adsorption of proteins to the capillary wall is that when using low pH background electrolyte, most of proteins are positively charged and the hydrophilic head of the fluorosurfactant is also positively charged. Under this condition, the proteins would be repelled from the capillary and certainly it is much more difficult for the proteins to be absorbed to the capillary inner wall. Meanwhile, fluorosurfactant molecules can interact with protein molecules. The hydrophilic head of the fluorosurfactant can interact with the hydrophilic region on proteins; or the alkyl chain of fluorosurfactant can interact with the hydrophobic region on proteins. Under both interactions, a ‘protection’ layer formed by fluorosurfactant is generated and it is much more difficult for proteins to be adsorbed to the capillary wall. (11) As a result, if low pH buffer is used, the adsorption of proteins to the capillary wall is highly decreased. (12)

1.4 Mass Spectrometry (MS)

Mass Spectrometry (MS) is a qualitative analytical technique which is used for the detection and analysis of the masses of analytes within a sample by measuring the charge‐mass ratios of the analytes. This technique has been widely used in chemical and biological research since it was developed. It is considered to be a powerful tool for protein separation and characterization to combine CE and MS.(13)

There are three main parts for every MS machine: an ion source, a mass analyzer and a detector. After the sample has been introduced to the machine, first with the help of the ion source, materials in the sample are ionized. There are many kinds of ion sources which have been developed and used in scientific research. However, the two most important and commonly used sorts of ion source for proteins are Matrix Assisted Laser Desorption/Ionization (MALDI) and Electro Spray Ionization (ESI). When the ionization step is finished, the mass analyzer will separate the ions. Two common ways of this separation step are Time of Flight (ToF) and Quadrupole. Ions are separated by the mass analyzer and then come to the detector, which is linked with a computer. The signals from the detector are transferred to the computer and processed. Last, the result of MS will be displayed on the computer. The working scheme of MS is shown in Fig 5. In this study, MALDI‐ToF was used for ionization and ion separation.

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Fig 5. Working scheme of Mass Spectrometry

MALDI is used for the analysis of large molecules like proteins, because it is a soft ionization technique, which affects the structure of sample material less than other ionization methods.(14) As the words ‘matrix assisted’ in MALDI say, MALDI needs some matrix materials to help to get the sample ionized. The sample for MALDI is mixed with a low molecular weight matrix material solution and applied on a metal MS plate before the ionization step forming crystals together. This matrix material can protect the analytes in the sample from being destroyed by the laser beam, which is used for ionization and keep the structure of the sample analytes intact. The matrix solution often contains trifluoroacetic acid (TFA), which can help the sample to get ionized. When the laser beam is shot upon the crystal of sample material and matrix, the energy of the beam is absorbed by the matrix and then transferred to the sample materials. In this way, the analyte molecules are ionized and then proceed to the mass analyzer.(15)

The most common type of mass analyzer combined with MALDI is Time of Flight (ToF). (16) Ions from MALDI are first accelerated in an electric field where they can receive a speed according to the charge they carry and the mass they have. Then they will enter a tube, where the time they take from entering the tube to reaching the detector is measured. This time is related to the charge‐mass ratio of the material, so that the molecular weight of the analyte can be easily measured.

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2 Materials and Methods

2.1 Materials

2.1.1 Samples and Reagents

The butterfly samples were acquired from Stockholm Universitetet (SU). All these butterflies were cultured manually and stuffed into specimens before transferred to the Royal Institute of Technology.

NaH2PO4, H3PO4 and dimethylsulfoxide (DMSO) were produced by MERCK company in Germany. NaOH pellet, trifluoroacetid acid (TFA), Ribonuclease A (RNase A), Lysozyme, and Bovine Serum Albumin (BSA) were manufactured by SIGMA‐AlDRICH company in USA. Acetonitrile (AcN) was made by Riedel‐de Haën company in Germany. 2,5‐Dihydroxybenzoic acid was produced by BRUKER company in Germany.

2.1.2 Equipments and Instruments

50/350 capillary and 50/150 capillary were manufactured by VITRO COM in UK.

Two sets of capillary electrophoresis instrumentations were used in this study. One was HEWLETT PACKARD 3DC with serial No. 3524900897 (will be called as HP commercial instrument in this thesis) and the other one is a homemade capillary electrophoresis instrument.

Fig 6. The HP commercial CE instrument (left) and the homemade CE instrument (right)

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The mass spectrometry instrument was BRUKER REFLEX with matrix assisted laser desorption/ionization and Time of Flight (MALDI‐ToF) detection.

Fig 7. BRUKER REFLEX with MALDI‐ToF detection

Vortex was IKA MS3 digital and the Centrifugator used was Biofuge pico from Heraeus company.

Fig 8. IKA MS3 digital (the blue one on the left) and Biofuge pico (the white one on the right)

The balance used was AND HR‐202. pH meter was 827 pH lab from Metrohm. The sonicator in this research was BANDELIN SONOREX RK100H. All water used in this research is Milli‐Q water obtained from the MQ water machine MILLIPORE Synergy 185.

2.2 Methods

2.2.1 Obtaining spermatophore from butterfly

Among the samples acquired from SU (Pieris Napi), very few female butterflies did not contain a spermatophore in their abdomens, while most of the samples have one spermatophore inside the bodies. To obtain the spermatophore, the dead butterfly sample was firstly placed on a piece of clean filter paper. The reason for using filter paper instead of

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common paper is that the filter paper is harder than common ones and it can absorb the tissues of the butterfly easily. The following step was to cut down the abdomen of the butterfly where the spermatophore is located. After the abdomen was separated from the other parts of the body, the spermatophore was pushed out from the abdomen using the backside of a knife with the help of a pair of tweezers. The spermatophore was then rolled on the filter paper by the backside of the knife in order to get rid of the tissues that were stick to the spermatophore. When this step was done, the color of the spermatophore was deep green and the surface of the spermatophore should be smooth. In addition, a tiny tooth‐like dark spot on it could be observed. Then the tweezers were used to stabilize the spermatophore while using the pin point of the knife to remove the spot carefully. Once the spot was removed, the last layer of tissue around the spermatophore left with it. At this moment, the spermatophore should be clean and in the color of white.

Fig 9. The procedure of obtaining butterfly spermatophore

2.2.2 Extraction of proteins from butterfly spermatophore

Since the contents of the spermatophore are not completely known, all the materials inside the spermatophore were obtained in this study. When performing this step, the spermatophore was first sliced inside a 1.5ml Eppendorf tube. Two to three spermatophores

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were used each time and 50µl MQ water per spermatophore was added after they were sliced. The tube was then applied to a vortex apparatus at the speed of 2500 rpm for 15min in order to suspend as much interior spermatophore materials as possible into the water. For the purpose of removing the tissues left, centrifugation at 3000 rpm for 10min was performed. The supernatant after the centrifugation was collected and the rest of the tissue parts were discarded.

The spermatophore protein samples were marked with the date when they were prepared and stored in 4� fridge. No spermatophore protein sample was stored for over 1 week since proteins got deteriorated after several days even if it was kept in the fridge. As a result, some electropherograms in Part 3 (Results and discussions) were obtained using different spermatophore protein samples.

2.2.3 Preparation of standard protein samples

Three kinds of standard protein sample solutions, Lysozyme, RNase A and BSA, were prepared for capillary electrophoresis. The concentrations of all these standard samples were 5mg/ml. Some features of Lysozyme, RNase A and BSA, which are related to the result of capillary electrophoresis analysis, are shown in Table 1 below.

Molecular Weight pI value Weight used in this research Lysozyme 14 kDa 11.1 5.02 mg RNase A 13.7 kDa 9.45 5.00 mg BSA 67 kDa 4.8 4.98 mg

Table 1. Some features of the standard proteins used in this research.

After weighing the protein sample powders using a balance, they were dissolved in 1 ml MQ water. These solutions were the standard samples for capillary electrophoresis.

2.2.4 Capillary Zone Electrophoresis (CZE)

The background electrolyte (BGE) was based on phosphate buffer made from NaH2PO4 and H3PO4 at pH 2.9.

(17) Three concentrations of buffer for CZE separation were prepared. They are 150mM, 100mM and 25mM. Another kind of buffer was made from 25mM phosphate buffer with 200µg/ml flourofactant.(18) Furthermore, two sets of CE machines were used as well. One was the HP commercial CE instrument and the other one was homemade CE instrument.

When using the HP commercial instrument, the length from the detection window to the outlet was not flexible. As a result, the total/effective length of the capillary was 60/52cm.

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150mM and 100mM buffer were used. The capillary used here was the 50/350µm capillary. The electrophoresis was carried out under 25kV voltage and 25�. Only the buffers without flourofactant were applied for this instrument.

Before the sample was injected, the capillary was flushed with 1M NaOH for 30min in order to let the silicon hydroxyl group on the inner wall of the capillary get dissociated into –SiO‐. This is called the activation of the capillary. Then the capillary was flushed with 0.1M NaOH, MQ water and the running buffer. Each flushing step took 30min. The capillary was first rinsed with 0.1M NaOH for 3min to flush out all materials in the capillary between two injections. Then MQ water and running buffer were applied for flushing so that the environment in the capillary could be restored to the running status.

For the homemade instrument, 25mM buffer with and without fluorosurfactant were used. The capillary used here was the 50/150µm capillary. For the buffer without fluorosurfactant, the total/effective length was 80/50cm and for the buffer with fluorosurfactant, the total/effective length was 130/100cm. 25kV voltage was kept the same while the temperature here was impossible to control.

When working with the buffer with fluorosurfactant, the flushing procedure was the same as the one for the HP commercial instrument before the first injection. However, after flushing with the running buffer, 25kV voltage was applied for 30min before injection. The reason for applying voltage was to stabilize the positively charged double layer of fluorosurfactant. Between each injection, the capillary was rinsed with only MQ water and running buffer, unless the capillary was jammed. 25kV voltage was applied for 10min as well between two injections.

All detection of CZE analysis with both sets of instruments were using UV light with 214nm wavelength, where the peptide bonds in proteins have their highest absorbance.

2.2.5 Mass Spectrometry with MALDI‐ToF detection

When performing MS, the 1µl protein samples from CE were mixed with 1µl 1mg/ml 2,5‐Dihydoxybenzoic acid which was used as matrix for MALDI.

At the same time, protein samples were also treated with ZiptipTM before mixing with matrix. A comparison between the sample pretreatments was performed as well. ZiptipTM is a kind of 10µl pipette tip which has C18 materials which can perform a solid phase protein extraction.(19) Before the samples were treated with Ziptip, several buffer solutions for this treatment needed to be prepared.

The wetting buffer was made from 100% AcN with 0.1% TFA. TFA was also dissolved in Milli‐Q water to reach the final concentration of 0.1% in order to make up the equilibration and washing buffer. The elution buffer contained 0.1% TFA and 50% AcN.

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When the protein samples were treated with ZiptipTM, the ZiptipTM was first put into the wetting buffer. The wetting buffer was aspired into the tip and then discarded for 10 times. Then the tip was equilibrated with the equilibration buffer in order to be able to bind the proteins. To fulfill this purpose, the tip was rinsed with the equilibration buffer 5 times. After this step, the tip was put into the protein sample and the sample was aspired and dispensed for 10 times. Then the tip with proteins bonded to it was washed with the washing buffer twice. Last, 4µl of elution buffer was used to elute the proteins out of the tip to a new Eppendorf tube. The buffer was also aspired and dispensed in the new tube for 10 times to avoid any possible protein loss. Then 1µl of the sample treated with ZiptipTM was mixed with 1µl 5mg/ml 2,5‐Dihydroxybenzoic acid on the MS plate.

When performing MALDI‐ToF detection of MS, each sample spot was analyzed with 20 laser shots and every spectrum was taken after 5 groups of shots, which means that every spectrum contains 100 shots. The laser intensity was set to 50% or 70%. The detection range was set to either 1‐5kDa or 1‐17kDa.

3 Results and Discussion

3.1 CZE using a buffer without fluorosurfactant

CZE was performed both on the HP commercial instrument and the homemade machine. Several conditions between these two machines were different and the electropherograms from two machines could not be directly compared, so results from the two machines are presented in two separate sections.

3.1.1 HP commercial instrument

When starting this research, the first set of conditions used was 150mM phosphate buffer without fluorosurfactant. This set of conditions was performed on the HP commercial instrument. In order to see whether this set of condition was working properly, several standard protein samples were applied to the CE instrument before the real butterfly sample. To mark the electroosmotic flow (EoF), dimethylsulfoxide (DMSO) was injected as well.

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Fig 10. CE result of DMSO, Lysozyme, RNase A and BSA

Fig 10 shows the result of the standard proteins and DMSO. In this figure, the blue line is the curve of DMSO, the red line is the curve of BSA, the green line is the curve of Lysozyme and the pink line is the curve of RNase A.

From this figure it can be seen that the peak of DMSO comes at 23 minute and the peaks of all protein samples come before the peak of DMSO. This means that under this set of conditions, all proteins are positively charged. Considering that the pI values of these proteins are all higher than 2.9, which is the pH of the buffer, this result is as expected. Moreover, since BSA has the highest molecular weight (Mw) and lowest pI value, the peak of it comes last among the protein samples. On the other hand, RNase A and Lysozyme have similar Mw but Lysozyme has a higher pI value, which means that Lysozyme may contain more positive charges under this set of condition. As a result, we see that the peak of Lysozyme comes earlier than the peak of RNase A. As a result, this series of experiments show that this set of conditions, including buffer conditions, voltage, injection and capillary conditions, can perform the task of detecting proteins successfully.

Following these experiments, the butterfly sample was analyzed. In figure 11, the result of this experiment is shown. The blue curve is the result of the butterfly sample while the red one is DMSO, which was used to mark the EoF.

Although it is difficult to say what conclusion can be exactly drawn from this electropherogram, some possible results can still be observed. For instance, we can see that most of the high peaks come before the EoF, which means a major part of proteins extracted from the spermatophore are positively charged. As a result, most proteins obtained from the spermatophore have pI values above 2.9. Meanwhile, still there are some peaks that come

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after the EoF. This means that there are also some proteins, which have the pI values below 2.9. As we know, very few proteins have such low pI values. So the environment within the spermatophore should be paid attention as these proteins can be interesting for further research.

Fig 11. The CE results from an analysis of butterfly sample ’0215’ and DMSO with 150mM buffer

After obtaining these results, the buffer concentration was changed to 100mM to see if there were any differences. Other conditions were not changed.

Fig 12. The result from an analysis of butterfly sample ‘0215’ and DMSO with 100mM buffer

Fig 12 shows the results of analysis of DMSO (red) and a butterfly sample (blue). The peak of DMSO comes at 14 minute, which is much earlier comparing with the one using 150mM

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buffer, which is shown clearly in Fig 13. The curve representing the butterfly sample has similar appearance with the one obtained using 150mM buffer but shows the same trend in relation to DMSO that all peaks come earlier. However, some noises and interferences can be seen in this curve after 25 minute. The reason for this noise and interference is unknown, but one possible reason would be that there is only one power route in the lab so that the current changes and interferences from other instruments in the lab could affect the current of this instrument.

Fig 13. The comparison of EoFs using different buffers

Fig 14. The comparison of the results of CE analysis of butterfly samples using different buffers

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In Fig 14, the red line is the curve obtained from analysis of the butterfly sample using 100mM buffer while the blue one is with 150mM buffer.

The first thing we can see in Fig 14 is that with 100mM buffer, the speed of EoF was increased, since all peaks came earlier. Furthermore, all peaks became sharper than the ones with 150mM buffer. As a result, the conclusion that decreasing the concentration of background electrolyte might result in the increase of the velocity of EoF and this result coincides with former research.(20) Moreover, 100mM buffer shows some tendencies that it may have better separation effect in this study comparing with 150mM buffer.

3.1.2 Homemade instrument

When using the homemade instrument, the concentration of buffer was decreased to 25mM and the capillary was changed to the 50/150µm type. The reason for changing capillary was that the 50/375µm one did not fit this machine well and the reason for decreasing the concentration so much was that when using 100mM and 150mM buffer, the current was too high (over 100µA). Since the capillary was much thinner, which means it is not as strong as the 50/375µm one, and the homemade instrument does not have any automatic cooling system, the capillary could not afford such high current and the heat generated from this current. When attempts with 50/150µm capillary and 100mM buffer were performed, the capillary almost got broken every time. Moreover, even if the capillary had survived the current and the heat, the electropherogram performance would be destroyed as well. As a result, the concentration of the buffer was decreased to 25mM to prevent the capillary from breaking down because of heat and current.

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Fig 15. The result of running DMSO with homemade instrument with 25mM buffer without fluorosurfactant

Since the length of the capillary was increased and the current was decreased a lot, the peak of EoF came much later, at 36 minute. Moreover, the curve looks much worse than the ones on the HP commercial instrument. There were lots of small peaks appearing all the time and peak of DMSO was wide and contained several small peaks as well. The reason for this could be that the injection volume was much higher. The pressure for injection on the homemade instrument was impossible to control under 500mbar, while the injection pressure on the HP commercial instrument was only 50mbar. High injection volume will cause large peak width and band spread. These would result in a bad electropherogram.

Fig 16. The result of analysis of a butterfly sample ‘0330’ and DMSO with 25mM buffer without fluorosurfactant

Fig 16 shows the CE results of DMSO (red) and a butterfly sample (green). Because different instruments were used, this result cannot be compared with the ones with 100mM and 150mM buffer. However, we all can see that although the electropherogram shows the same trend as the one obtained with 100mM and 150mM buffer, it is much worse than those. According to these results, the conclusion was that this set of conditions did not work well with the homemade instrument, because the homemade instrument could not provide a good environment such as temperature control and injection control. This would result in many uncertainties and cause the bad result. As a result, the condition would be changed again.

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3.2 CZE using a buffer with fluorosurfactant

To improve the separation performance, fluorosurfactant was added into the buffer. Since the fluorosurfactant used in this study has a positively charged hydrophilic head, the EoF is reversed if positive potential is still applied to the inlet end of capillary. As a result, to keep the direction of EoF towards the outlet end, the electrodes need to be reversed as well. This means that the negative potential was applied to the inlet end and positive charge to the outlet end. The main purpose of adding fluorosurfactant is to decrease the adsorption.

Fig 17. CE result of analysis of DMSO and butterfly sample ‘0517’ with 25mM buffer with fluorosurfactant

Fig 17 shows the CE result of running DMSO (red) and a butterfly sample (green). After adding fluorosurfactant into the buffer, several changes appeared. The first one was that the baseline became straight. The main reason for this could be that adsorption was decreased.(21) The second change was that the EoF was faster than without fluorosurfactant, which can be observed in Fig 18.

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Fig 18. Comparison of DMSO electropherograms using two different buffers

Fig 19. Comparison of butterfly sample electropherograms using two different buffers

The third difference is that, all peaks are sharper and clearer than those obtained without fluorosurfactant. It was discovered that all peaks obtained with the BGE with fluorosurfactant were smaller than those in the BGE without fluorosurfactant. This can be observed in Fig 19. There are a couple of possible reasons for this phenomenon and the main reason was that it was too difficult to make sure that every injection had the same volume without an automatic injector. It was also possible that the absorbance of BGE was changed after adding fluorosurfactant, which would affect the height of the peaks as well.

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Anyway, adding fluorosurfactant was actually altering the whole background electrolyte system, and the charge carried on the inner capillary wall was totally different with the BGE without fluorosurfactant. Thus, it is very difficult to analyze which factors were involved in the change of the velocity of EoF and the height of peaks. Further results are required to determine the reason for these differences.

3.3 MS

The first step here was to compare the MS of samples treated with ZiptipTM and without. The result is shown in Fig 20.

The butterfly sample ‘0607’ was used here and it was before the separation with CZE. The detection range was 1‐5kDa and the laser intensity was 70%. As we can see, the sample which was not treated with ZiptipTM showed a very bad MS result. The noise was too high and the protein signals were too low. Peaks of proteins can hardly be observed. On the other hand, after the treatment with ZiptipTM, the peaks of proteins were high and clear.

The reason for why proteins treated with ZiptipTM gave a much better MS result could be that ZiptipTM provided a solid phase extraction, which means that the proteins got purified again by the ZiptipTM.(22) Moreover, when performing analysis of the fractionated samples, some materials in the CZE buffer could cause chemical interference in the MALDI analysis and ZiptipTM reduced this. Although some proteins might be lost during the treatment with ZiptipTM, the loss could hardly be observed. In addition, even if there was no loss of proteins without the treatment, nothing could be seen if the sample was not treated. So it was considered that the treatment with ZiptipTM was necessary before the sample was applied to MS and as a result, all protein samples was treated with ZiptipTM in further research.

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Fig 20. MS results of sample treated with Ziptip (above) and without (below)

In accordance with the electropherograms obtained using CZE with fluorosurfactant, the whole sample was fractionated into 3 fractions. An Eppendorf tube was put at the outlet end of CZE when the CZE run was started and the materials were collected for 13 min before changing to another tube. Then the materials were collected for another 13 min. In this way, 3 fractions were collected. After the treatment with ZiptipTM, they were made into MS samples and applied to MALDI‐ToF analysis. The detection range was 1‐5kDa and the laser intensity was 70%. The sample used here is the butterfly sample ‘0607’. The result is shown in Fig 21.

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Fig 21. The MS result of butterfly sample (fraction 1 to 3 from left to right, respectively)

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To compare the spectra from the three fractions, they were put together and the result is shown in Fig 22.

Here we can see, a strange phenomenon had occurred. There are some peaks which appeared in all three spectra, which means that the same proteins existed in all three fractions. It is difficult to say what caused this to happen. Meanwhile, as we can see, there are also some peaks which showed up in only one fraction. This means that some kinds of proteins specifically appeared in only one fraction. The outlet of the capillary was also cleaned before it was put into a new Eppendorf tube when collecting the fractions to avoid cross contamination.

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Fig 22. Comparison of three fractions of sample ‘0607’

To determine if this was caused by the butterfly sample, another butterfly sample ‘w53’ was used. All conditions of MS was kept the same and the result is shown in Fig 23.

However, as we can see, this picture shows almost the same trend as with Fig 22. Still some kinds of proteins showed up in all three fractions, although some other proteins were specific to only one fraction. From this result, we can only say that it was not the butterfly sample which caused this consequence. As a result, it is impossible to explain why this would happen with these results and the method to solve this phenomenon is still not known at this moment.

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Fig 23. Comparison of fractions of butterfly sample ‘w53’

The MS analysis of the butterfly samples was also performed with a method optimized for the range 1‐17kDa. The laser intensity was 60%. The result is shown in Fig 24. There are two huge peaks which can be observed, which refers to that at least two kinds of proteins whose molecular weights are higher than 5kDa exist in the butterfly spermatophore. However, this result shows the same trends as was shown for the range 1‐5kDa as these two proteins appear in all three fractions.

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Fig 24. Comparison of fractions of butterfly sample ‘0607’ under 1‐17kDa

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Last, a comparison between two different butterfly samples was performed. This analysis was made using the same method which was optimized for the range 1‐5kDa and the laser intensity was 70%. The result is shown in Fig 25.

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Fig 25. Comparison between butterfly sample ‘w53’ (above) and ‘0607’ (below)

These results shows some interesting points. We can see that most proteins in these two samples are the same. Meanwhile, there are also some proteins specific to only one sample. This gives us a hint that most proteins extracted from the butterfly spermatophore are the same for every individual, and we can suppose that they are necessary for every butterfly to keep the speramtophore to work correctly. However, there must be some differences between individuals. Because the purpose of this research is to study the method for all male butterflies to prevent the females from mating with other males, the proteins that appeared in both samples need to be paid more attention than the specific ones. This does not mean that those proteins are not important, because they show the specificity of every individual and the complexity of the species. But for further research on this topic, those ones are less important.

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4 Conclusions

Since this research includes a series of steps from obtaining the spermatophore to characterization of the proteins, lots of experiments have been done. As a result, several conclusions could be drawn. The important conclusions are mainly related with the separation and analysis of the proteins. They are described into the following parts separately.

4.1 Optimizing the conditions for capillary electrophoresis

First, it was proved that butterfly spermatophore proteins could be separated with the phosphate buffer without fluorosurfactant. The types of proteins which could be separated and detected are almost as many as the one under the buffer with fluorosurfactant. The phenomenon that supports this hypothesis is that numbers of peaks which could be observed from both methods are almost the same. Meanwhile, it is discovered that for the buffer without fluorosurfactant, when the concentration of the buffer is decreased, the EoF becomes faster. 100mM buffer gave faster and better separation effect than 150mM buffer for butterfly spermatophore proteins. However, the adsorption of proteins to the capillary wall could not be eliminated under this set of conditions. The baseline was not straight and the noise was very high.

Then, it is observed that adding 200µg/ml fluorosurfactant to CZE buffer with 2.9pH can result in even faster separation and low protein adsorption to capillary wall. Moreover, this set of conditions gave better repeatability. But since there were so many restrictions when working with the homemade machine, such as no temperature control and automatic injection, the conditions might be further improved with better sets of instruments.

4.2 Conclusions from MS results

Although the fractionation step in the MS part was not so successful, still some conclusions can be drawn. The first conclusion is clear and obvious. That is, there are many kinds of proteins existing in butterfly spermatophores. In addition, it was observed that most proteins in different spermatophores are the same. These reproduction proteins might be important for the research of the pathway that male butterflies use to prevent the female ones from mating with other males. At the same time, it was observed that there were some

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differences in the sorts of proteins between individuals. This might be interesting but less important for further research on this specific topic.

Furthermore, it was discovered that some proteins peaks which existed in the whole sample got lost after fractionation, because the proteins in the sample were actually diluted again during the fractionating process, so that the final concentrations in the fractions were below the detection limit of MS and this resulted in the loss of some peaks. The method to eliminate this drawback of fractionating needs to be developed.

4.3 Further research

Since a procedure for obtaining spermatophores and extracting proteins from the spermatophore has been well established in this study, further research needs to be focused on optimizing the methods for protein separation and analysis. As we can see, although the proteins can be separated by CE very well, and the electropherogram looks good, there might be some possibilities that the conditions still can be improved. Faster and better separation will always be the aim for CE method research.

On the other hand, the problem for MS that every fraction show the same kind of proteins needs to be addressed as well. This problem can be caused by the CE analysis, but it is also possible that something went wrong during the sample preparation for MS. Moreover, the sample preparation method might be improved as well.

When all the separation and analysis conditions are settled and the proteins are well separated and detected, further work like the determination of the proteins’ structures can be performed. However, that will be a huge project and would not be finished very soon. Anyway, this would be the direction of this whole project, and this research is just a cornerstone and a start point of it.

To summarize, it could be concluded that as a first initial study on butterfly reproduction proteins, this project has fulfilled its duty and the work has gone on well, although there are still several details which are not proved to be optimized. The basic idea of this research was to keep as many parameters stable as possible and only very few ones were variable. Under the lead of this plan, the separation of proteins using capillary zone electrophoresis showed good results and the conditions have been generally optimized. These results also coincide well with former research(23). All this suggests that this research has gone in the correct direction and the results obtained were promising. At the same time, as a totally unknown area, the analysis of those proteins using mass spectrometry has provided several fundamental results. Although those results are very simple and basic, they can be used as both a comparison template and step stone for future work.

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As a result, a whole process from obtaining a spermatophore to the analysis of the proteins has been primarily developed from this work, and several important clues of the butterfly spermatophore proteins have been received. With the optimization of the separation conditions and improvement of detection and analysis strategy, more and more information on butterfly reproduction proteins will be obtained.

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5 List of Abbreviations

AcN Acetonitrile

BGE Background Electrolyte

BSA Bovine Serum Albumin

CE Capillary Electrophoresis

CZE Capillary Zone Electrophoresis

DMSO Dimethylsulfoxide

DNA Deoxyribonucleic Acid

EoF Electroosmotic Flow

HPLC High performance liquid chromatography

kDa Kilo Dalton

MALDI Matrix Assisted Laser Desorption/Ionization

MQ water Milli‐Q water

MS Mass Spectrometry

Mw Molecular weight

pI Isoelectric point

RNase A Ribonuclease A

TFA Trifluoroacetic acid

ToF Time of Flight

UV Ultra Violet

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6 Acknowledgements

First of all, I would like to thank Åsa for giving me the fantastic chance to perform this interesting and exciting topic. Thank you for all you advice and suggestions for my work. I have learnt so many things from this topic.

Meanwhile, I want to thank Maria, Patrik, Inma and other colleagues for your kind help and advice on my work. Especially I want to thank Johan for all your kind help during my work. I couldn’t have spent a wonderful time here in the Division of Analytical Chemistry without you.

And also I wish to thank all my friends. It is impossible for me to finish this work so smoothly without all your support and help.

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7 References

1. Haerty, W., Jagadeeshan, et, al. Evolution in the fast lane: rapidly evolving sex‐and reproduction‐related genes in Drosophila species. Genetics, 177 (2007): 1321‐35.

2. Adams E.M. and Wolfner, M.F. Seminal proteins, but not sperm, induce morphological changes in the Drosophila melanogaster female reproductive tract during sperm storage. J. Ins. Physiol. 53 (2007): 319‐31.

3. K‐G. Heller, S. Faltin, P. Fleischmann, O.v. Helversen. The chemical composition of the spermatophore in some species of phaneropterid bushcrickets (Orthoptera: Tettigonioidea). Journal of Insect Physiology, 44 (1998): 1001‐1008.

4. Geoffrey D. Findlay, Willie J. Swanson. Proteomics enhances evolutionary and functional analysis of reproductive proteins. BioEssays, 32 (2010): 26–36.

5. E. Santoyo, R. Garcia, K.A. Galicia‐Alanis, et, al. Separation and quantification of lanthanides in synthetic standards by capillary electrophoresis: A new experimental evidence of the systematic ’odd–even’ pattern observed in sensitivities and detection limits. Journal of Chromatography A, 1449 (2007): 12‐19.

6. Armstrong D W, Schulte G. Separating Microbes in the Manner of Molecules in Capillary Electrokinetic Approaches. Anal. Chem, 71 (1999): 5465‐5469.

7. Okun V M. Analysis of Common Cold Virus (Human Rhinovirus Serotype 2) by Capillary Zone Electrophoresis: The Problem of Peak Identification. Anal. Chem, 71 (1999): 2028‐2032.

8. Li‐Yao Zhang, Xing‐Chun Tang, Meng‐Xiang Sun. Simultaneous determination of histamine and polyamines by capillary zone electrophoresis with 4‐fluor‐7‐nitro‐2,1,3‐benzoxadiazole derivatization and fluorescence detection. Journal of Chromatography B, 820 (2005): 211–219.

9. Pier Giorgio Righetti, Cecilia Gelfi. Recent advances in capillary zone electrophoresis of DNA. Forensic Science International, 92 (1998): 239–250.

10. Jörg Hanrieder, Aida Zuberovic, Jonas Bergquist. Surface modified capillary electrophoresis combined with in solution isoelectric focusing and MALDI‐TOF/TOF MS: A gel‐free multidimensional electrophoresis approach for proteomic profiling—Exemplified on human follicular fluid. Journal of Chromatography A, 1216 (2009): 3621–3628.

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11. Jean‐Louis Salager. Surfactants‐‐Types and Uses. Laboratory of Formulation, Interfaces Rheology and Processes. University of Los Andes. (2002).

12. Åsa Emmer, Johan Roeraade. Wall deactivation with fluorosurfactant for capillary electrophoresis analysis of biomolecules. Electrophoresis, 22 (2001): 660‐665.

13. Mark E. McCom, Andrew N. Krutchinsky, Werner Ens, et, al. Sensitive high‐resolution analysis of biological molecules by capillary zone electrophoresis coupled with reflecting time‐of‐flight mass spectrometry. Journal of Chromatography A, 800 (1998): 1–11.

14. Rob Haselberg, Gerhadus J. de Jung, Govert W. Somsen. Capillary electrophoresis‐mass spectrometry for the analysis of intact proteins 2007‐2010. Electrophoresis, 32 (2011), 66‐82.

15. Karas M, Bachmann D, Hillenkamp F. Influence of the Wavelength in High‐Irradiance Ultraviolet Laser Desorption Mass Spectrometry of Organic Molecules. Anal. Chem. 57 (1985): 2935–2944.

16. Brandon T. Ruotolo, Kent J. Gillig, Earle G. Stone, et al. Analysis of protein mixtures by matrix‐assisted laser desorption ionization‐ion mobility‐orthogonal‐time‐of‐flight mass spectrometry. International Journal of Mass Spectrometry, 219 (2002): 253–267.

17. Tereza Varilova, Hana Semenkova, et al. Affinity liquid chromatography and capillary electrophoresis of seminal plasma proteins. J. Sep. Sci, 29 (2006): 1110 – 1115.

18. Åsa Emmer, Mårten Jansson, Johan Roeraade. Improved capillary zone electrophoretic separation of basic proteins, using a fluorosurfactant buffer additive. Journal of Chromatography A, 547 (1991): 544‐550.

19. Li‐Ming Wei, Qian Shen, Hao‐Jie Lu, Peng‐Yuan Yang. Pretreatment of low‐abundance peptides on detonation nanodiamond for direct analysis by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry. Journal of Chromatography B, 877 (2009): 3631–3637.

20. J. L. Beckers, F. M. Everaerts, M. T. Ackermans. Determination of absolute mobilities, pK values and separation numbers by capillary zone electrophoresis: Effective mobility as a parameter for screening. Journal of Chromatography A, 537 (1991): 407‐428.

21. Å. Emmer, M. Jansson, J. Roeraade. Separation of pig liver esterase isoenzymes and subunits by capillary zone electrophoresis in the presence of fluorinated surfactants. Journal of Chromatography A, 672 (1994): 231‐236.

22. Doris E. Terry, Edward Umstot, Dominic M. Desiderio. Optimized Sample‐Processing Time and Peptide Recovery for the Mass Spectrometric Analysis of Protein Digests. American Society for Mass Spectrometry, 15 (2004): 784–794.

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23. Anne Varenne, Stephanie Descroix. Recent strategies to improve resolution in capillary electrophoresis—a review. Analytical chemistry, 628 (2008): 9‐23.

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