Compositional Analysis of Spider Silk

Supervisor: Dr. Brian Kelleher

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Abstract
The chemical composition of spider silk is currently under intensive investigation due to its impressive mechanical properties such as tensile strength and elasticity. The protein content of the fibers has received a lot of attention, whereas other smaller components of the silk have not yet been studied as thoroughly. Lipids and low molecular weight compounds have been reported on the webs of some spiders, however little is known about them. The following report documents the chemical composition of spider webs, with additional focus on the lipids present, as well as reporting the findings of the silk analysis performed throughout the study.

Contents
Chapter 1. Introduction........................................................................................6 1.1 Spider Silk..............................................................................6 1.2 Silk Production.......................................................................7 1.3Composition of the Silk...........................................................8 1.3.1 Silk Proteins.............................................................8 1.3.2 Silk Lipids................................................................9 1.3.3 Lipid Function........................................................11 Chapter 2. Gas Chromatography-Mass Spectrometry Analysis of Silk..............13 2.1 Introduction...........................................................................13 2.2 Materials and Methods..........................................................16 2.3 Results and Discussion..........................................................19 Chapter 3. Silk Analysis using Scanning Electron Microscopy...........................28 3.1Introduction............................................................................28 3.2 Materials and Methods..........................................................31 3.3 Results and Discussion..........................................................32 Chapter 4. Silk Analysis using Infrared Spectroscopy........................................38 4.1Introduction............................................................................38 4.2 Materials and Methods..........................................................40 4.3 Results and Discussion..........................................................41 Chapter 5. Conclusions and Further work...........................................................45 5.1 Conclusion.............................................................................45 5.2 Further work..........................................................................46 References...........................................................................................................47 Appendix.............................................................................................................50

Chapter 1. Introduction
1.1 Spider silk
Silk is a remarkable biomaterial which has interested humans for thousands of years.(1-3) It is a natural protein fibre which can be produced by several species.(4) These include many varieties of insects, most notably, silkworms, caterpillars, webspinners, ants, bees and wasps. Other types of arthropods, such as arachnids, also produce silk. Spiders are the largest order of arachnids.(5) The production of silk by spiders is one of the most well known sources of silk in the environment. Additionally, they are the only animal to utilize silk in their daily life.(6)

The chemical composition of silk and the silk-spinning process is currently under intensive study due to its impressive mechanical properties such as tensile strength and elasticity. These properties are unmatched in the natural world and are comparable with the best synthetic fibres produced by industries.(2)(5)(7-10)On a weight-to-weight basis, spider silk is approximately five times stronger that high tensile steel.(11) Spider silk also displays an average tensile strength three times greater that of the silkworm Bombyx mori and is approximately ten times higher than other natural materials such as collagen, chitin and cellulose. (12) In terms of elasticity, certain types of spider silk can extend by almost 300%.(2) These properties are required to absorb the impact energy of prey onto the web and prevent the web from breaking. Spider silk is also very resilient and quite acidic which protects it from micro-organisms. (2)(5) It is spun at near ambient temperatures and pressures, with water as a solvent, producing an environmentally safe, biodegradable material.(11)(13-15) This, in addition with high tensile strength and elasticity, has made spider silk very attractive for commercial applications. (1)(2) While insect silk is primarily been used in the textile industry, spider silk has been utilised for many different applications. It has been used as crossed hairs in optical instruments, as fishing nets and for wound dressing. (2)(5)

At present, biomedical application of silk materials is under review. The high elasticity, biodegrability, antithrombic nature and low inflammatory potential of silk proteins are attractive qualities for a biomaterial in this area. Surgical thread, degradable sutures, artificial tedons and ligaments for knee construction and scaffolds for tissue engineering are some of the possible areas of application. (1)(4)(15-16) The use of spider silk in a body armour system and for ballistic protection is another area of interest.(14) This is because the silk fibers of the web have the ability to stop prey mid-flight and convert their momentum to heat energy. 70% of the converted energy is dissipated and the web rebounds so the prey is not catapulted back out. This dissipation of energy at very high strain rates makes spider silk ideal for this application. (7)(12)

However, obtaining the required amount of silk for these applications poses many problems. It is time-consuming, highly expensive and since most spiders are cannibals, domestication of these arthropods to create spider farms for silk production is impractical.(2)(11) Therefore, scientists have been studying the chemical composition and spinning process of spider silk in an attempt to clone silk genes for overexpression in bacteria, yeast and mammalian cell culture systems and produce recombinant silk proteins.(1)(12) These recombinant silk proteins could be produced in large quantities have the desirable mechanical properties and features of natural spider silk for commercial applications.(2) (11)

1.2 Silk Production

Currently there are over 37,000 species of spider known(11), all of which produce silk.(17-18) Some spiders are capable of secreting up to seven different types of silk that vary in biological function.
(11-13)(19-20)

The different types of silk are all primarily protein based

polymers.(21) The silk is produced by spinnerets, a spiders silk-spinning organ. Each spider has two to eight sets of spinnerets, with six sets being the most common. These are located on the underside of a spiders abdomen. Numerous silk glands open into the spinnerets. The silk is secreted from the spinnerets, through the glands, in liquid form. It then hardens into a solid thread due to a drop in pH and the application of strain. (22) Each gland produces a different type of silk. The variety in silk type has evolved and been produced to accommodate for the

multitude of function of a spiders silk. (5)(11)Diverse material is required for web design, radial threads, prey capture, immobilizing prey, egg casings and ballooning.(1-2)(4)(17)(19) Table 1. Types of Silks, Glands and Their Functions. Adapted from Saravanan (2006). (12) Silk Dragline Viscid (Flag) Glue-like Minor Cocoon Wrapping Attachment Gland Major Ampullate Flagelliform Aggregate Minor Ampullate Cylindrical Aciniform Piriform Spinneret used Anterior/ Median Posterior Posterior Anterior/ Median Anterior/ Posterior Anterior/ Posterior Anterior Function Orb web frame, radii Prey capture, sticky spiral Prey capture, attachment Orb web frame Reproduction Wrapping captured prey Attachment

1.3 Composition of the silk 1.3.1 Silk Proteins

The primary constituents of spider silk are complex protein molecules.(2) Amino acid composition, sequence and various end groups in spider silk have been studied in great detail.
(7)

It has been discovered that the main amino acids present consist of alanine, glycine, serine,

glutamine, tyrosine, leucine, valine and proline. In comparison, silk produced by silk worms contains the first three amino acids only. (5)

Silk proteins, unlike functional protein, are not comprised of one conservative sequence. They consist of unknown non-conservative segments and repetitive conservative sequences which vary according to silk type.(13) These repetitive sequences can sometimes account for over 90% of the spider silk protein.(2) They are short polypeptide stretches, 10-50 amino acids in length, which can be repeated over one hundred times within a single protein.(2) Each polypeptide has distinct functional features which attribute to the high elasticity and tensile strength of spider silk threads.

DNA sequence analysis of the different types of silk, from different species, has revealed the polypeptide stretches to be common protein architecture. They can be grouped into four 8

categories; (i) (Gly-Ala)n/An (ii) Gly-Gly-X (iii) Gly-Pro-Gly-X-X and (iv) spacers which contain charged amino acids.(2)(11-12) The amino acid X may be glutamine, tyrosine or leucine.(5)(23) These polypeptide stretches are present in all silk types except those from the aggregate and piriform glands which have yet to be determined.(24)

These amino acid sequences fold in different manners. Analysis in this area has revealed that the polypeptide stretch (Gly-Ala)n/An will tend to form -sheet structures in assembled fibres and -helices in solution. (25) These regions form randomly orientated crystallites which have been stated to be responsible for the high tensile strength on spider silk.
(9)(11)

On the other

hand, the structures acquired by Gly-Gly-X and Gly-Pro-Gly-X-X have not yet been identified. Literature in this area has suggested two possible structural formations for these sequences, amorphous rubber-like structures (26) or the formation of a 31-helical structure. (25) The rest of the silk protein consists of non-conservative, non-repetitive sequences. These nonrepetitive regions are located at the proteins termini and are important in the assembly of silk proteins into fibres.(2)(9) They are approximately 100-200 amino acids long and demonstrate well defined secondary and tertiary structures in solution. The carboxy-terminal nonrepetitive sequences have been identified for several different silks and spiders. It has revealed a high sequence homology amongst these domains. (2)

1.3.2 Silk Lipids

The presence of lipids has recently been discovered on spider silk. Lipids, of different compound classes were found on the webs of Linyphia triangularis (Linyphiidae) and other spiders by accident.(5) The primary components of these silk lipids are unbranched, oddnumbered alkanes and even-numbered 2-methylalkanes, such as 2-methyloctasane. They are also often accompanied by fatty acids, including palmitic, stearic and oleic acid, alkanols, alkanediols, glyceryl ethers as well as small amounts of aldehydes, ketones, fatty acid amides and wax-type esters.(5)(12)(27-28)

It is still unknown on which type of silk the lipids are located. They have been found present in low abundances on dragline silk from Nephila species. However, these lipids are absent on the silk from hunting spiders, such as Cupiennius salei, which only produce dragline silk. It

has therefore been concluded that it is likely that the lipids are associated with one of the other types of silk or are applied to silk after the web has been constructed. (27)

Figure 1. Lipid components, including 1-methoxy-2,22-dimethylpentacosane, 1-methoxy-2(methoxymethyl)-12-methylnonacosane, 1-methoxy-16,20,24-trimethylhentriacontane and ester icosyl 2,4,6-trimethyltridecanoate, from different spiders. Adapted from Schulz et al. (1997) (5)

In addition to these compounds, methyl-branched alkyl methyl ethers are present on the spider silk. These methyl ethers (1-methoxyalkanes), unlike the compounds mentioned above, are unique to spiders and have only been identified so far in spider silk.(29) They can vary in chain length from 23 to 37 carbon atoms. More than 100 ether-containing lipid compounds from 15 species have been identified at present.(5)(27)

The lipids on the webs of Nephila Clavipes, and other Nephila species, can be composed of up to 80% of 1-methoxyalkanes.(28) This 80% is dominated by internally branched ethers such as 1-methoxy-16,20,24-trimethylhentriacontane. These had long chain lengths of C28 to C34 with up to four methyl groups in the chain. The methyl groups are separated by three methylene units, as often found in anthropod hydrocarbons. They are located close to the alkane end of the molecule and are mostly arranged in a 1,5- pattern. 1-methoxy-2,28dimethyltriacontane is the only compound with a methyl group at the C2 position.(27) The second largest group of lipids reported by Schulz et al.(1999)(27)were hydrocarbons, predominately even-numbered 2-methylalkanes. Unlike the 1-methoxyalkanes, the

hydrocarbons were in a variety of structural conformations and included branched compounds. They were also present as mono-, di-, tri- and tetramethyl compounds. As the 10

majority of the hydrocarbons are even-numbered alkanes, it is unlikely that Nephila Clavipes uptake hydrocarbons in their diet. This is because the major alkanes of flies are oddnumbered hydrocarbons.(27)

Lipid analysis was performed on the webs of 10 different spiders of the Linyphiid species by Schulz et al (1999)(27). It was determined that the majority of the lipids present were hydrocarbons, and between 5% and 40% were ethers. The compositions of the hydrocarbons did not vary to a great extent between species. Pentacosane, heptacosane, 2methylhexacosane, 2-methyltriacontane and 2-methyloctacosane were shown to be the most abundant compounds. (27)

The lipid layer on the silk of Pholcus phlangoides (Pholcidae), also known as the daddy-longlegs spider, does not consist of a complex mixture. It is composed of up to 90% of the branched wax type ester icosyl 2,4,6-trimethyltridecanoate. The rest of the lipid layer consists of small amounts of homologs, hydrocarbons, aldehydes and methylketones.(5)(27)

1.3.3 Lipid Function

The function of the lipids on spider silk is unknown. There have been many possibilities suggested.(5)(9)(12)(27-28)(30) One function may be protection of the thread from environmental influences.(5)(9)

Figure 2. Schematic diagram of the composite structure of a MA fiber. Taken from Hardy et al (2010) (7)

As can be seen above, the lipid layer resides on the outside of the silk. Consequentially, any chemical or biological agent has to penetrate the lipid barrier before reaching the core of the fiber. It has widely been supposed that the protein of spider silk is chemically protected against microorganisms and it is thought that the presence of lipids may contribute to 11

this.(9)(27) 14-methylhexadecanoic acid, 12-methyltetradecanic acid and other similar acids are found on spider silk and are known to possess high antimicrobial activity. (5)(12) Another function for silk lipids may be communication(27) or species recognition amongst spiders. Sex pheromones are imbedded into a specific lipid matrix on the webs of female spiders. Attracted males come into contact with them. It is thought that these males may recognise correct webs by pattern recognition of this matrix.(30)

Indication of the use of silk lipids for species recognition can be seen from the species Argyrodes trigonum, from the family Theridiidae, which live cleptoparasitic in the webs of F. Pyramitela. A. trigonum will sometimes consume their host but will never attack each other. However, when the lipids are removed by organic solvents, this kind of cannibalism occurs.(5) Regulation of water content on the web is another suggested function for lipids.(5)(12)(27) The cuticle of a spider, and other arthropods, is covered by a lipid layer. This lipid layer regulates the water balance of these arthropods. Aliphatic hydrocarbons, fatty acids and small amounts of waxy esters, aliphatic alcohols and cholesterol are the primary components of this cuticle lipid layer. As previously discussed, similar compounds exist on the silk. Optimal water balance in the web is required for it to function properly. The water content can help in holding the silk taut and contribute to its elastic properties which are needed for good prey capture. It has been suggested that the lipid layer on silk reduces the evaporation and uptake of water. In addition to this, it is thought that the methyl branches of the lipids promote an enhanced mobility of the components in the lipid layer thereby aiding in the protection of the thread during sudden elongation.(5)

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Chapter 2. Gas Chromatography - Mass Spectrometry Analysis of Spider Silk

2.1 Introduction
Gas Chromatography, coupled to Mass Spectrometry, is the most common technique used in the compositional analysis of silk lipids.(28-29)(31) In general, only a few micrograms of lipid material is extractable from each web.(5) Gas Chromatography-Mass Spectrometry (GCMS) can be used to resolve and identify the components contained within this small quantity of material, which can contain up to 150 different compounds. n-Alkanes, branched alkanes, methoxyalkanes, fatty acids, alcohols, sterols, sugars, carboxylic acids, aldehydes and wax esters are among some of the compounds which have been identified in spider silk using GCMS. The combination of reference libraries, extracted ion chromatograms of diagnostic ions, common mass spectra fragmentation patterns, gas chromatographic retention times, injected standards and chemical derivitizations can all be extremely beneficial in the characterization of these components too. The first detailed characterization of silk lipids was published by Schulz (2001). (28) Extracts of silk from Nephila Clavipes was studied using GCMS. This analysis revealed the presence of hydrocarbons, alcohols and unique methyl-branched alkyl methyl ethers. The total lipid content was estimated to range between 3-5% of the dry fiber.

The results showed that the methyl ethers were long chain (C28 to C34) and made up 50-80% of the lipids. They were identified by characteristic ions at m/z 45, which corresponded to CH3OCH2, and loss of m/z 32 from the molecular ion, which corresponded to loss of methanol. The second largest lipid group were the alkanes. These were found to predominately contain methyl branchings with the major components being 2methyloctacosane and 2-methyltriacontane. Dimethyl, trimethyl and tetramethyl, odd and even numbered alkanes, as well as n-alkanes were also detected. (28)

Analysis of methoxyalkanes, and also alcohols, can sometimes prove difficult as their spectra can be very similar. Methyl branches located in the middle of the molecule or close to the ether linkage has proven difficult identify unequivocally. This can sometimes be attributed to 13

how easily water or methanol is lost from the molecular ion when using mass spectrometry. When determining branching positions, methyl ester branches between C 2-C4 are relatively easy to identify due to the high electron density from carbonyl induced fragmentations. However, identification of branching at other positions is more difficult to determine due to their low ion intensities. The use of retention indices and chemical derivitization is often the greatest help in the determination of the amount methyl groups located along the methyl ethers and, indeed, their branching positions.(29)

In the study of silk lipids of Nephila Clavipes, the methyl ethers were transformed into cyanides with trimethylsilyl iodide followed by treatment with tetraethylammonium cyanide in CH2CL2 at room temperature.(28)

Figure 3. Gas chromatograms of silk lipid extract. Adapted from Schulz (2001).(28)

Figure 3 shows some of the gas chromatograms produced during this analysis. The first chromatogram shows a crude extract and the second shows an extract that was derivatized with cyanides prior to analysis. Better resolution can be seen in the second chromatogram due to the later elution of the cyanides. This enabled the identification of minor hydrocarbons that had previously coeluted with some of the methyl ethers. Furthermore, the results of the mass spectra concluded that the ethers contain up to four methyl branches. (28)

Partial characterization of the silk lipids of Linyphia Triangularis was also obtained by Schulz et al. (1993) using GCMS.(31) Methyl branched methoxyalkanes were identified using 14

a combination of gas chromatographic retention indices, characteristic fragment ions and chemical modifications. These compounds comprised 30% of the lipid extract and consisted of 27 different variations. The chain length of these compounds was found to vary between C24 and C32 with multiple methyl branches.(31) In this study, retention indices were used successfully to distinguish between mono-, di- and trimethyl methoxyalkanes. The positions of the branches were then identified using derivitization methods. The methoxyalkanes were converted from methyl ethers to methyl esters, using RuO4, which exhibited a different fragmentation pattern. They were also converted to hydrocarbons through transformation into iodides using trimethylsilyl iodide and reduction with lithium aluminium hydride. These derivitization steps produced different mass spectroscopic fragmentation patterns and permitted the determination of branching positions. Reference compounds, of both the methyl ethers and methyl esters, were also synthesized to aid the verification of results.(31) Chemical derivatization can also be performed using nitriles.(29)The methyl ethers and alcohols in the extracts are converted to their respective iodides, as previously done, but are then reacted with Et4NCN in dichloromethane, producing nitriles. Characterization of methyl branching positions via nitriles allows the production of very characteristic mass spectra with easily identifiable functional group positions, as well as the production of a clean derivatized sample which is almost free of impurities. Fatty acids and their esters may also be converted into alcohols and analysed in this manner too.(29)

GCMS has been the dominant technique used in the identification of silk lipids to date. It is also the standard method of choice for the separation and identification of lipids and their fatty acid components in general. This is due to its ability to perform a complete quantitative analysis of the lipid composition of the sample in a relatively short period of time. The only technique comparable with GCMS for this type of analysis is HPLC in reverse-phase mode. Although HPLC operates at an ambient temperature and has been used for isolating specific components and for radioactivity measurements, the running cost is much higher and the identification of the sample components can be difficult. The choice of detector is also of concern as most lipids lack chromophores or fluorophores for spectrophotometric detection. Light-scattering detectors and MS detectors are becoming more common although they have yet to be used in the investigation of silk lipids. 15

2.2 Materials and Methods

Spider Maintenance and Silk Collection

Tegenaria spp. was collected from a domestic residence in north Dublin in early spring. The female spider was housed alone in a 57 x 27 x 33cm aquarium tank. The aquarium tank was lined with gravel at the bottom. Multiple twigs and stones, which had been thoroughly washed with deionised water, were inserted into the tank to facilitate web construction. The tank was stored in a bright, well-ventilated location at room temperature. The spider was fed small crickets once a week. Feeding did not take place during web production to reduce the possibility of contaminating the silk with lipids from the insect prey. Water was offered daily from soaked tissue paper.

Silk was collected every morning onto a sterile, alcohol washed glass rod. The webs were collected by cutting the scaffolding threads with a clean scalpel (washed with methanol between samples), and twirling the silk around the top of the rod. Rods were stored upright in a small tube, under a clean inverted graduated cylinder.

Silk was also collected from webs found around Dublin City University using the same sampling method. This silk material was pooled together and used for preliminary lipid analysis.

Extraction of Free Lipids

The silk was scraped off the rod, using a clean scalpel, into a Teflon tube and freeze-dried. The sample was then sonicated for 15min with 10 mL HPLC grade methanol, then 10 mL dichloromethane:methanol (1:1; v/v) and finally with 10 mL dichloromethane. The solvent extracts were combined and filtered through glass fiber filters (Whatman GF/A). The extract was concentrated by rotary evaporation, transferred to a 2 mL chromatography vial and dried completely under a steady stream of nitrogen gas. The solvent extract was then redissolved in 500 L dichloromethane:methanol (1:1; v/v) and aliquots were taken for derivatization. The

16

remaining web material, containing non-extractable material, was air-dried and stored in the fridge at 5OC.

Fractionation of extracts

The extracted material was separated into 3 fractions, neutral lipids, polyhydroxyalkanoates (PHAs) and polar lipids, using solid-phase extraction (SPE). Discovery

DSC-18, 50mg/1

mL, SPE tubes and Supelco Ultrapure aminopropyl, 500 mg/3 mL, SPE tubes were used . The cartridges were conditioned with 1 mL of chloroform, 1 mL of acetone, 1 mL of HPLC grade methanol, 1 mL of 4% acetic acid in diethyl ether and 1 mL of HPLC grade hexane. The flow rate was controlled to allow the reagents to pass slowly through the column and to prevent the column from drying out. The flow rate was reduced by approximately half the speed to load the sample. 200 L of sample was allowed drain through before washing the cartridge with 200 L of HPLC grade hexane. After the hexane had passed through, a clean glass vial was under the cartridge to begin collecting. 1 mL of chloroform was added and the neutral lipids fraction was collected. A new vial was placed under the cartridge and 1 mL of acetone was added to collect the PHA fraction. The vial was replaced again and the final fraction, the polar lipids, was collected with the addition of 1 mL of HPLC grade methanol. The cartridge was allowed to drain completely to dryness into this vial. Fractions were then derivatized as required.

Derivatization of natural extracts

Silylation via N,O-bis-(trimethylsilyl)trifluroacetamide (BSTFA) and pyridine The alcohols and acids present in the silk were converted to trimethylsilyl (TMS) derivatives through the replacement of the active hydrogen atoms by a TMS group. A 100 L aliquot of the extracted material was transferred to a 2 mL glass vial and completely dried under nitrogen. The extract was then derivatized by reaction with 90 L BSTFA and 10 L pyridine. The vial was placed into an oven at 70OC for 3 hours. After cooling, 100 L HPLC grade hexane was added to dilute the extract before injection onto the GC-MS.

Esterification using boron trifluoride (BF3) and silylation with BSTFA and pyridine This two-stage derivitization process involved the production of fatty acid methyl esters and TMS ethers through acid-catalysed esterification and base-catalysed silylation. A 100 17

L aliquot of the extracted material was transferred to a 2ml glass vial and completely dried under nitrogen. 50 L of the esterification reagent, BF3/methanol solution (14% w/w), was added. 50 L of chloroform was added to aid solubility. The solution was vortexed and heated in the oven at 70OC for 45 minutes. 450 L of deionised water was added to terminate the reaction. The methyl ester products were extracted with 1 mL of HPLC grade hexane and chloroform [9:1]. The mixture was shaken vigorously and the upper organic layer was retained. This process was repeated again and the combined BF3 extracts were dried over sodium sulphate and evaporated to dryness under nitrogen. 50 L chloroform, 45 L of BSTFA and 5 L of pyridine were added to the residue and the solution was vortexed. The reaction was maintained at 70OC in the oven for 2 hours. Excess reagents were evaporated under nitrogen gas and the resulting residue was reconstituted in 100 L of HPLC grade hexane and chloroform [9:1] before injection onto the GC-MS.

Analysis The webs collected around Dublin City University, the Tegenaria lipid extract and the derivatized SPE fractions of the extract were analysed using capillary gas chromatographymass spectrometry. The system used was a Varian 450-GC gas chromatograph, coupled to a Varian Saturn 2000 series ion trap GC/MS system. The GC system contained a Varian 1177 split/splitless injector with SGE Focusliner set at 2500C. A split ratio of 20:1 was used. The MS system was operated in electron impact (EI) mode at 70eV with helium as the carrier gas. It scanned from m/z 40-650. Separations were performed on an Agilent J&W Scientific DB5ms, 30 m x 0.25 mm, film thickness 0.25 m. The oven temperature was programmed to hold at 750C for 2 mins, then increase from 75-3000C at 60C/min and finally to remain at 3000C for 20 mins, creating a total run time of 59.50 mins.

18

2.3 Results and Discussion

Preliminary Lipid Analysis

Preliminary lipid analysis was performed on silk that was collected and pooled together from many webs. This extract produced the following chromatogram.

Figure 4. GC-MS chromatogram of the pooled sample.

The results identified the primary lipid classes to be fatty acids, mono-unsaturated fatty acids, sugars, including monosaccharides and disaccharides, alkanols and n-alkanes.

19

Table 2. Sugars present in the pooled sample. Sugar Monosaccharide Retention Time 26.488 26.892 28.661 28.786 30.121 31.396 26.488 Disaccharide 40.567 40.984 41.416 Table 3. Fatty Acids present in the pooled sample. Retention Time 21.772 28.955 32.039 33.446 34.523 34.849 36.144 37.422 38.647 39.824 44.817 48.409 Compound Name Decanoic acid Tetradecanoic acid Hexadecanoic acid Heptadecanoic acid Octadecenoic acid Octadecanoic acid Nonadecanoic acid Eicosanoic acid Henicosanoic acid Docosanoic acid Hexacosanoic acid Octacosanoic acid Lipid Number 10:0 14:0 16:0 17:0 18:0 18:1 19:0 20:0 21:0 22:0 26:0 28:0 Compound Name Levoglucosan Levoglucosan Fructose Fructose Glucose Glucose Levoglucosan Sucrose Maltose Maltose

Table 4. Alkanols present in the pooled sample. Retention Time 33.604 30.706 27.528 Compound Name 1-octadecanol 1-hexadecanol 1-heptadecanol

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Table 5. Alkanes present in the pooled sample. Retention Time 34.203 35.561 36.865 38.126 41.648 42.867 44.232 45.806 Compound Name Docosane Tricosane Tetracosane Pentacosane Octacosane Nonacosane Triacontane Hentriacontane C22 C23 C24 C25 C28 C29 C30 C31

Table 6. Other compounds identified in pooled sample. Retention Time 12.063 17.816 25.239 39.430 49.143 Compound Name Propanoic acid Phosphoric acid Triethylamine Monopalmitin Palmityl palmitate

As can be seen from figure 4, the majority of the compounds in the sample were identified. They were identified through a combination of the use of reference libraries, such as Wiley and NIST, injected standards, common fragmentation patterns and extracted ion chromatograms of diagnostic ions. The use of relative retention times was also a brilliant asset in the identification of the long chain aliphatic compounds such as the alkanes, alkanols and fatty acids. The importance and versatility of GC-MS as a separation and identification technique was clearly evident by the amount of compounds that were identified and the number of techniques that can be used in conjunction with it to aid identification.

The choice of extraction solvents and derivatization techniques were also seen to yield good results. Alcohols have proven to be good extraction solvents for most lipids, with methanol 21

and ethanol being the most commonly used. Chloroform is also a popular solvent. It has good extraction capabilities for lipids of intermediate polarity when mixed with methanol. However, it is not very stable. Dichloromethane has similar properties to it but is less oxidizable. This is a very beneficial trait for a solvent to have in lipid analysis as lipids are prone to autoxidation. If polyunsaturated fatty acids are not protected they will autoxidize very rapidly. This can also happen in air, which is why the samples were dried down under nitrogen during sample preparation.

The samples were derivatized to reduce their polarity and to decrease the possibility of the analytes forming hydrogen bonds with the capillary column. In doing so, the analytes were better separated, thereby improving peak shape and resolution simultaneously. BF3/methanol converted the fatty acids to fatty acid methyl esters through acid-catalysed methylation and the resulting reaction with BSTFA silylated the hydroxyl groups of the sterols and aliphatic alcohols creating trimethylsilyl ethers. This assisted in detection also by the production of characteristic fragmentation patterns for these compounds.

The unlabelled peaks in figure 4 are either contaminants, such as siloxanes or phthalates, or compounds that were also in the control sample. Contamination from siloxanes and phthalates generally originates from plastics. This is likely to have been introduced from the use of plastic gloves, plastic pipettes or from plastic vial caps during sample preparation. Fortunately, these sources of contamination are readily identifiable. Phthalates have a characteristic base peak at m/z 147 and the siloxanes have a common fragmentation pattern when found in their cyclic structure. A way of reducing this contamination would be to avoid the use of plastics and instead to use Teflon lined caps and glass pipettes.

All of the compounds that were found would be typical in most environmental samples, including aerosols, sediments and soils. However, as the sample was of many different webs pooled together it is impossible to say which species produced them. Additionally, as they were collected from the environment, there may be some unknown sources of contamination or lipids that were introduced from external sources. It is also likely that some unsaturated fatty acids may have been lost due to autoxidation in the atmosphere. As a result, it was decided to obtain a single web-producing spider for the silk lipid analysis instead. This spider could be identified and kept in a controlled environment, thereby producing fresh clean webs for analysis. 22

Results of Tegenaria extract

Tegenaria spp. A female spider was obtained from a domestic residence in north Dublin. She was identified as a Tegenaria gigantea or Tegenaria saeva due to her size and markings, by the British Arachnological Society. She had an approximate leg span of 5 cm and a body length of 1.5 cm. These two species are identical in size and appearance and hybridize as well, making them difficult to tell apart. Based on Irish distribution data, the spider is more likely to be a Tegenaria saeva. However, this could be made more certain by studying the female epigyne of the spider or through DNA testing.

Both T. saeva and T. gigantea can live indoors or outdoors. They produce large sheet webs to capture their prey, often with a tubular corner for them to retreat. These webs are very durable and if abandoned, are sometimes inhabited by spiders of other species.(32)

Both species reach maturity in two years. They are typically born in late spring and mature the following year, males in July/August and females in September/October. Mating then begins in early autumn with the males searching for the females. After choosing their mate, the male will guard the female until she undergoes her final moult and reaches maturity. Afterwards, the spiders will co-habit on the females web, mating repeatedly until the male dies. The female then stores the sperm and produces a succession of egg sacs the following spring. They can produce 10 or more egg sacs, depending on food supply, each containing 40-60 eggs. The females usually die the next winter. (32)

During the course of the project, the Tegenaria spider produced eggs 4 times. This indicated that it was a female spider. The eggs were pale yellow and spherical. They were contained in a sac hanging from the web.

23

Figure 5. Images that were captured of the Tegenaria spider and used for identification.

Figure 6. GC-MS chromatogram of the Tegenaria extract.

24

Figure 6 shows the GC-MS results of the Tegenaria extract. In comparison with the chromatogram of the pooled web sample, there were fewer peaks present. This was expected as it is from the silk of only one species and was produced in a clean, controlled environment. However, a lot of the same lipid classes were present, including fatty acids, alkanes, alkanols and sugars. This suggests little variation in compound type between species.

The individual compounds were also quantified using cholestane as an internal standard. Cholestane is a steroid hydrocarbon that does not occur naturally in the environment. As it is very non-polar, it is retained well by the column and doesnt elute until high temperatures are reached (>300OC). Consequently, it is less unlikely to co-elute with any sample peaks or to mask any peaks.

A series of standard addition calibration plots, one for each lipid class, were obtained from a reliable source, and as per standard procedure, the equation of the lines produced were used to calculate the concentrations of the individual compounds.

Table 7. Sugars identified in Tegenaria silk and their associated quantities.

Retention [S] (ug Total -1 time Compound Peak Area [As/Ais] [Cs/Cis] mL ) mass (ug) ug mg-1 26.493 Leuvoglucosan 18428 3.61E-05 0.038875 3.887536 0.272128 0.14 26.848 Leuvoglucosan 175409 0.000343 0.039005 3.90049 0.273034 0.14 28.632 Fructose 2850533 0.005581 0.041212 4.121238 0.288487 0.14 28.756 Fructose 1109682 0.002173 0.039776 3.977585 0.278431 0.14 30.091 31.362 40.535 Glucose Glucose alpha DGlucopyranoside 10053929 8355862 5774315 0.019685 0.01636 0.011306 0.047157 0.045755 0.043625 4.715654 4.575532 4.362505 0.330096 0.320287 0.305375 0.17 0.16 0.15

Table 8. Sugars identified in Tegenaria silk and their associated quantities.

Retention time 30.68 27.499

Compound 1-hexadecanol 1-heptadecanol

Peak Area 897355 178865

[As/Ais] 0.001757 0.00035

[Cs/Cis] 0.024037 0.022897

[S] (ug mL-1) 2.403709 2.289691

Total mass (ug) 0.16826 0.160278

ug mg-1 0.08 0.08

25

Table 9. Alkanes identified in Tegenaria silk and their associated quantities. Retention [S] (ug Total time Compound Peak Area [As/Ais] [Cs/Cis] mL-1) mass (ug) ug mg-1 32.76 Heneicosane 1413534 0.002768 0.026594 2.659445 0.186161 0.09 34.178 Docosane 2045960 0.004006 0.02818 2.818012 0.197261 0.10 35.539 Tricosane 3091499 0.006053 0.030802 3.080158 0.215611 0.11 36.849 Tetracosane 4902193 0.009598 0.035341 3.53415 0.24739 0.12 38.104 Pentacosane 5067518 0.009922 0.035756 3.575601 0.250292 0.13 39.315 Hexacosane 5763247 0.011284 0.0375 3.75004 0.262503 0.13 40.49 Heptacosane 5102044 0.009989 0.035843 3.584258 0.250898 0.13 41.631 Octacosane 3429999 0.006716 0.03165 3.16503 0.221552 0.11 44.218 Triacontane 2561385 0.005015 0.029472 2.947244 0.206307 0.10 45.788 Hentriacontane 1763071 0.003452 0.027471 2.747084 0.192296 0.10 Table 10. Fatty Acids identified in Tegenaria silk and their associated quantities. Retention time 25.551 28.929 31.647 31.999 33.424 34.481 34.807 [S] (ug mL-1) 6.273038 7.448801 6.643296 14.5579 6.220037 7.200549 10.05826 6.089154 6.373406 9.327609 Total mass (ug) ug mg-1 0.439113 0.521416 0.465031 1.019053 0.435403 0.504038 0.704079 0.426241 0.446138 0.652933 0.22 0.26 0.23 0.51 0.22 0.25 0.35 0.21 0.22 0.33

Compound Dodecanoic acid Tetradecanoic acid Hexadecenoic acid Hexadecanoic acid Heptadecanoic acid Octadecenoic acid Octadecanoic acid 12:0 14:0 16:1 16:0 17:0 18:1 18:0 20:0 24:0

Peak Area 1197828 5759917 2634468 33343985 992180 4796673 15884916 484337 1587265 13049895

[As/Ais] 0.002345 0.011278 0.005158 0.065285 0.001943 0.009392 0.031102 0.000948 0.003108 0.025551

[Cs/Cis] 0.06273 0.074488 0.066433 0.145579 0.0622 0.072005 0.100583 0.060892 0.063734 0.093276

37.4 Eicosanoic acid 42.121 Tetracosanoic acid 36.906 Hexanedioic acid

Table 11. Other compounds that were identified but not quantified

Retention time 36.906 47.592

Compound Hexanedioic acid Cholesterol

Peak Area 13049895 3106690

None of the classes of silk lipids have yet been quantified in published material. This makes it impossible to compare these values against other spider species. This is new information. Quantification of the results showed the fatty acids and sugars to be the most abundant compounds in Tegenaria saeva silk. A list of fatty acids, alkanols and sugars found in spider silk has also not been published.

26

However, as previously mentioned, the silk lipids of Nephila Clavipes and Linyphia Triangularis have been characterized.
(28-29)(31)

Both these species reported the presence of

methoxyalkanes and of branched alkanes being the most abundant compounds. Neither of these types of compounds were identified in the silk of the Tegenaria spider.

Solid phase extraction (SPE) was also performed during the course of this study. This was carried out to separate the lipids into three fractions; neutral lipids, polar lipids and a PHA fraction. Ideally this would make the chromatograms clearer, improve resolution, and make identification easier. SPE is performed regularly in lipid analysis but has not yet been implemented in silk lipid analysis. It was performed three times during the project but yielded no significant peaks. It is most likely that this was because an insufficient amount of sample was used and more sample would be needed. However, as there was only one spider, silk was produced slowly and there was not enough time in the course of the project to collect a larger sample of silk and repeat the analysis again. The performance of SPE is potentially very beneficial to this kind of analysis and shouldnt be disregarded as a valuable technique in this area.

More chromatograms of pooled silk samples, Tegenaria silk samples and SPE samples are available for viewing in the appendix of this report.

27

Chapter 3. Silk Analysis using Scanning Electron Microscopy

3.1 Introduction

Scanning Electron Microscopy (SEM) is an analytical technique that uses a high energy beam of electrons to produce a high quality image of the surface of the sample. It can be utilised to provide an insight into the orientation and conformation of silk strands.(33) The electrons interact with the atoms on the surface of the sample and provide information about the samples composition, topography and electrical conductivity by examining objects on a micrometer to nanometer scale. It has been used for a wide variety of applications in silk analysis in the past, including studying structures in the silk, examining silk thread diameter and investigating the hierarchical structure silk. (10)(15-16)(21-22)(33-34) In a study by Bielecki (2009) (21) the silk structures of a range of spider species, from seven genera, were examined using SEM. The samples were prepared by sputter coating them with gold and viewing them at magnifications up to 35,000x. Images were produced that showed several types of silk structure that were common among the species, such as large rounded bilateral fibres with an approximate diameter of 1.29 m and strands with equidistant glue droplets which varied in size among species. The common occurrence and high abundance of these features suggests that they may contribute significantly to web functionality.(21)

Figure 7. 2a. Salticidae Rounded bilateral strand (front), 2b. Cryphoeca Rectangular bilateral strand, 2c. Cryphoeca Rounded bilateral strand, 2d. Mimetus Equidistant glue droplets, 2e. 28

Mimetus Higher magnification of glue droplets, 2g. Mimetus Droplets on a rounded bilateral strand with unusual droplet (left). Adapted from Bielecki (2009) (21)

The presence of other structures, which are shown below, including ridged, corkscrew, semibraided, single and multiple stranded threads were also reported as being present in some of the silk samples. It was deduced that these types of strands were only produced by species that use these structures for species specific functions. Possible functions of the structures were assigned but it was stated that further study would be required to confirm them. The high resolution images that were produced using SEM permitted identification of the structures and differentiation between them, illustrating clearly one application of SEM. (21)

Figure 8. 2h. Salticidae Unusual branched structure, 2i. Cryphoeca Ridged fibre, 2j. Salticidae Corkscrew fibres,2k. Salticidae Wound bilateral fibre, 2l. Salticidae Semi-braided fibre, 2m. Salticidae Unusual structure. Adapted from Bielecki (2009). (21)

A large variation in silk strand diameter was also detectable using SEM. A number of studies have reported a range of strand diameters, increasing from 100 nm up to 11.5 m.(15)(21) There was no consistency seen between strand structure or type of strand and its size. Using SEM, it was observed that the common rounded bilateral strand varied from 110 nm to 3.84 m in diameter and the glue droplets varied substantially from 220 nm to 43.75 m. (21)

It has been suggested that the amount of spider silk protein present affects the diameter. In a study by Zhang et al. (2010), spider silk protein was electrospun with poly-L-lactic acid to form a composite nano-fiber for biomedical application.(15) It was discovered that the diameter of the composite fiber decreased significantly as the amount of silk protein increased, suggesting a possible relationship between protein content and strand diameter.(15) 29

The use of Energy Dispersive X-ray (EDX) analysis, in conjunction with SEM, has proven itself to be a valuable technique in silk analysis additionally. As x-rays are emitted from deep within the sample, they can provide excellent compositional information about the sample for elemental analysis or chemical characterization. Similarly to SEM, EDX has been used for a broad range of applications.(16)(22) In a paper by Yang et al.(2010)(16), calcium phosphate was deposited onto recombinant spider silk fibres to help improve bone repair and regeneration for use in biomedical applications. EDX was utilised in this study to follow the mineralization process. A series of x-ray spectra were recorded at different time intervals during the coating process to investigate the dynamics of coating formation. Ca, P, Na and Cl were mapped allowing the determination of which elements distributed homogenously or if they were more concentrated in certain areas.

Figure 9. SEM micrograph of a cross-sectioned coated fibre and its corresponding EDX spectrum. Adapted from Yang et al. (2010). (16)

When the sample was cross-sectioned, EDX was also used to measure the thickness of the coating. The study shows that EDX can be used for qualitative and quantitative analysis.(16) Knight et al (2001)(22) used EDX analysis to investigate the changes in the elemental composition of the silk dope of Nephila edulis as it travels through the spinning duct and exits the spigot forming a thread. The abundances of sodium, chlorine and potassium were measured and plotted against distance along the duct. It was found that the concentration of Na+ and Cl- decreased progressively along the duct, whereas K+ increased. It was deduced that this, accompanied by a drop in pH, helps the protein molecules to refold and crystallize as they exit the spider, showing control of the ionic environment in the duct is necessary in forming the thread and another application of EDX. (22) 30

3.2 Materials and Methods

Sample Preparation

Both fresh silk and previously extracted silk from the Tegenaria spp. was prepared for analysis by mounting it on a sticky carbon tab which was adhered to a 15mm aluminium stub. Carbon paste was also used sometimes instead of a carbon tab. The aluminium stub, carbon tab and carbon paste were used due to their electrically conductive nature. All samples were sputter coated with gold for 4 min. This was to prevent the build-up of charge, as well as increasing electron signal and surface resolution as the emission of electrons near the surface increases, as silk is a non-conductive sample. The samples were then ready for insertion into the SEM.

Analysis

Fresh silk was analysed using a Hitachi S-3000N SEM with Hitachi S-3000 software. The accelerating voltage ranged from 3-4 kV as it was a biological sample. The other operating conditions included a probe current of 28-35, a working distance of 5-6mm and an aperture setting of 3 or 4 for imaging purposes. The images were taken at a range of magnifications up to 25,000x.

Both the fresh and extracted silk were used for EDX analysis. These were analysed using a Hitachi S3400N SEM with. The accelerating voltage was set at 3.5kV. The other operating conditions included a probe current of 70, a working distance of 15mm and aperture setting 1, as these samples were examined for analysis purposes.

31

3.3 Results and Discussion

SEM

Silk Structures As previously stated, some spiders are capable of secreting up to seven different types of silk which are primarily composed of different configurations of proteins, each engineered for a specific function. During this analysis, images were generated that showed a variety of silk structures and the presence of different types of silk. These images were then compared with the structures that have already been published in literature to help in assigning possible functions.

10a

10b

Figure 10. 10a. Rounded bilateral strand (right), possible multiple stranded fibre (left), 10b. Aqueous glue droplets.

Figure 10 shows the presence of a rounded bilateral strand, with a diameter of 732 nm, and of silk glue droplets. These were the only two structures that were present in all seven genera of spiders tested by Bielecki (2009) (21). Their common presence indicates that these two types of silk are essential for web functionality. It is therefore likely that they correspond to major ampullate (Ma) silk and flagelliform (Flag) viscid silk respectively.

Both MA and Flag silks contribute to the elasticity of the web and are good energy absorbing materials needed for efficient prey capture.
(1)(2)(26)

MA silk is used for formation of the

supporting frame, radii and hub of the web. It is possible then that multiple stranded fibres, 32

such as in figure 10a, provide further support to the web. Flag silk forms the adhesive capture spiral. The Flag silk itself is not sticky. It has been postulated that the web receives its adhesiveness via spider glue proteins which can be seen above. These glue droplets are manufactured by the aggregate gland of the spider and are coated on the outside of the strand as it emerges from the spinneret.(5)(11)(35) During this analysis, a large difference in the size of the glue droplets was clearly evident, making it difficult to say if they are spaced equidistantly from each other as has been reported in literature.(21)

11a

11b

11c

11d

Figure 11. Series of images of a frequently occurring unknown strand.

Another type of silk was observed frequently during imaging. This silk, shown in figure 11, was moderately sized, with a diameter averaging 770 nm. It contained circular raised structures on its surface. It is possible that these structures are smaller glue droplets and these strands are a form of Flag silk but as these strands arent reported in literature and SEM is an imaging technique it is impossible to state for certain the type or function of this variety of silk without further testing on it.

33

Other structures were present but appeared less commonly in the silk. These structures included ridged strands and semi-braided fibres and are shown below in figure 12. Ridged strands appeared in the silk of 3 genera out of the 7 tested by Bielecki (2009) (21) and semibraided fibres appeared only twice. It is thought that each species of spider only produces the types of silk which relate to the tasks employed by their species, implying that Tegenaria spiders utilize these structures for specific functions. It has been suggested that the ridged fibres contribute strength to the structure of the web and the semi-braded strands improve the elasticity as they are visibly loose and could be stretched upon stress. (21)

12a

12b

12c

12d

Figure 12. 12a. Ridged fibre, 12b. Semi-braided fibre, 12c. Minor ampullate silk, 12d. Crosssection of a silk strand.

Minor ampullate silk is visible on the right hand side of image 12c. This is single stranded silk which is used to form the non-adhesive auxiliary spiral which stabilizes the body of the web. Image 12d shows the cross-section of a silk strand, possibly minor ampullate silk. The protein contained within the strand is visible but the image is not clear enough to observe the lipid coating. This could possibly be due to how the strand was broken. If EDX analysis was performed on a strand such as this, the lipid content should be detectable and its thickness could be measured by mapping the elemental composition across the top of the strand. 34

Silk Diameter A variation in the diameter of the silk was also visible under SEM. Table 12 below shows the average size of the various types of silk and the diameter range, if observed multiple times, for the Tegeneria spider.

Table 12. Silk diameter sizes Silk Type Single stranded Multiple stranded Rounded bilateral Glue-containing Ridged Semi-braided Unknown silk with raised structures Diameter Range (m) 0.32-0.47 0.92-2.58 0.73-0.88 0.20-2.7 0.77-0.78 Average Diameter (m) 0.37 1.81 0.80 1.5 1.63 0.98 0.77

The multiple stranded fibres, glue-containing strands and ridged fibres were found to be the largest types of silk. It is unlikely that the diameter of the different silk types has a notable effect on the performance of the individual strand but is more likely related to the amount of protein molecules present. The different types of silk in the web affect its performance collectively and diverse material is required for the necessary attributes for good prey capture, such as tensile strength and elasticity.

EDX analysis EDX analysis was performed on fresh silk and on extracted silk with the aim of investigating the elemental composition of each and see was there a difference between the two. Fresh silk was obtained from the spider just before analysis and was quickly sputter coated with gold. As this silk was not pre-treated with any solvents or exposed to the atmosphere for a long period of time, it should theoretically still have a completely intact lipid layer on the outside of each strand. Therefore, the predicted detectable elements for this sample included carbon, hydrogen and oxygen as these are the primary components of all lipids.

35

Figure 13. SEM image of area selected for EDX analysis and its corresponding x-ray spectrum.

The area for analysis was firstly chosen using SEM. It was decided that area analysis as opposed to spot analysis would provide results that were more representative of the entire sample. The x-ray spectrum, provided above, showed the presence of carbon, nitrogen, oxygen and three peaks for gold. The gold was introduced via the sample preparation and the three peaks are due to the different amounts of energy are released by the various shells in the atom. Hydrogen was also not detected because the diameters of the orbitals are too small.

The presence of nitrogen, however, was unexpected. It is possible that the small amount of nitrogen discovered originated from glycoprotein within the aqueous glue droplets which are also composed of water and low molecular mass compounds. As these glue droplets are on the surface of some strands, it is quite likely that their composition could slightly influence the elements detected and their relative abundances.

There is also a small possibility that the nitrogen originated from the protein in the sample as EDX is not a surface technique like SEM as x-rays are generated from approximately 2 microns in depth. This would depend on both the thickness of the gold coating and the thickness of the glue droplets and the lipid layer making it very unlikely. This increases the probability that the nitrogen is from the glue droplets.

36

Figure 14. SEM image of the extracted silk and its corresponding x-ray spectrum

Figure 14 above shows the x-ray spectrum generated for the extracted silk. This silk had previously been extracted for GCMS analysis, air-dried and stored in the fridge at 5OC, before being prepared for analysis. Theoretically, the lipid coat on the silk should have been removed due to this procedure and the complex protein molecules contained inside should now be visible for detection. As protein is composed of carbon, hydrogen, oxygen and nitrogen, these elements were expected to be observed. A noticeable increase in the amount of nitrogen was also expected subsequently.

Although there was a small increase in the level of nitrogen, it was not significant enough to say that there was a notable difference between the two types of sample without further testing with a larger sample sizes. A potential reason for only a small increase could be that the extraction process was not fully efficient and some of the lipid layer remains, although, this is unlikely due to the wide range of compounds that were detected by GCMS.

It is concluded that the analysis should be repeated with more samples to increase confidence in there being a notable difference being the two sample types. This would also mean that one could tell if they were looking at the lipid layer or at the protein content. A more efficient method of doing this would be to cryomicrotome the silk samples so the cross-section of the strands could be studied. EDX could be then used to map the change in elemental composition across the top of the strands. It could be seen easily if there was a difference in composition, or nitrogen levels, between the edges of the strands and the core of the fibre. However, this equipment was not available for use during this project.

The full EDX spectra available for viewing in the appendix of this report.

37

Chapter 4. Silk Analysis using Infrared Spectroscopy

4.1 Introduction
Infrared Spectroscopy (IR) is one of the most common spectroscopic techniques used in gathering information about a compounds structure, assessing purity and sometimes in the identification of compounds. In terms of silk analysis, FT-IR spectroscopy provides information about the secondary structure of the protein. Characteristic bands, known as Amide I and Amide II, result from the amide bonds that link amino acids. Amide I is primarily associated with stretching vibrations of the C=O of the amide and Amide II with the bending vibrations of the N-H bond. As these bonds are involved in the hydrogen bonding that occurs within the secondary structure, the locations of these bands are sensitive and strongly affected by the structure and folding of the protein. (15)

Due to the anisotropic nature of fibres such as silk however, analysis using this technique may be difficult.
(36)

Additionally, the majority of the silks properties are related to their

degree of orientation parallel to the axis of their fibre which poses problems when using vibrational spectroscopic techniques. Consequently, a variety of sampling methods have been tested and compared in a brilliant paper by Van Nimmen et al (2008)(36) in an attempt to overcome these technical difficulties. KBr-disk technique, transmission microscopy, attenuated total reflection IR (ATR-IR) and diffuse reflectance infrared spectroscopy (DRIFTS) were all implemented in the study of egg sac fibres of Araneus diadematus.

Figure 15. FT-IR results for egg sac spider silk. Taken from Van Nimmen et al (2008). (36) 38

As shown in figure 15 above, the different techniques produced results that varied from each other, especially in the region above 1500 cm-1, for the same sample type. KBr-discs and transmission microscopy were determined to be less suitable modes of FT-IR analysis of silk as resolution was poor in the Amide I region. This is more than likely affected by the particular geometry of the fibres. Due the adhesive nature of silk, it is also very difficult to cut the threads smaller than 100m for preparation of a uniform disc. ATR-IR and DRIFTS provided more information about the sample, had minimum sample preparation and were the most repeatable of the techniques. However, a slight shift of absorption peaks to lower wavenumbers was observed for ATR-IR and a slight shift to higher wavenumbers was seen with DRIFTS. It was deduced that these shifts were a result of the specific nature of the techniques, although they were repeatable and well-resolved, particularly DRIFTS. (36)

IR has been used for a variety of applications in silk analysis, although noticeably less than both GCMS and SEM/EDX. In the study by Zhang et al. (2010),(15) mentioned previously, IR was used in this analysis for investigating the molecular conformation of the electrospun fibres. The amount of silk protein was varied in the fibres and this variation was detectable by IR. By studying the increase or decrease in characteristic absorption peaks, the effect of the silk protein content on secondary protein structure could be determined. As the protein content was increased, two characteristic peaks occurred at 1653cm-1 and 1546cm-1 corresponding to -helix and random coil of silk protein. Bands between 1610cm-1 and 1641cm-1 were assigned to -sheet conformation. IR showed that the composite fibres had more -helix structures than electrospun spider silk by itself.(15) IR was also used in a study by Ene et al (2010) (37) to investigate the conformational changes in the silk of Nephila edulis after it had been hydrogenated and partially deuterated. This analysis was interesting as it looked at the structural changes of the protein structure when the silk had supercontracted. Supercontraction is the term used to describe the dramatic shrinking of silk fibers by up to 50% in response to hydration.(1)(5)(38)Frequency shifts were observed, as well as peaks changing in size and a new peak appearing at ~1750cm-1. These conformational changes are particularly important as they affect the mechanical properties of the silk.(37)

IR was beneficial in both these analyses for detecting conformational changes in silk structure. It has proven itself to be a valuable technique, particularly in the investigation of silk secondary structure, provided the limitations of the various modes of IR are considered. 39

4.2 Materials and Methods

FT-IR analysis was performed on silk collected from the Tegenaria spider. It was carried out using two modes of IR, attenuated total reflection FT-IR (ATR FT-IR) spectroscopy and preparation of a Nujol mull.

ATR FT-IR spectroscopy ATR FT-IR spectroscopy uses a property of total internal reflection for the direct measurement of solid or liquid samples. It does not require any sample preparation. It was performed using three days worth of silk production (3 webs, <1 mg of silk) on a Perkin Elmer Spectrum 100 FT-IR Spectrometer with Universal ATR and FT-IR Microscope Attachment. The measurements were performed at room temperature (21OC). The sample was scanned 4 times with wavenumbers range 4000-650cm-1. The background spectrum was subtracted from the sample spectra.

Nujol Mull Nujol oil (liquid paraffin) is a saturated hydrocarbon which is commonly used as a dispersant for an organic compound. The silk was combined with Nujol, using a pestle and mortar, to make a thick suspension or mull. Two weeks worth of silk production (c. 2 mg of silk) was required to produce bands which were differentiable from Nujol peaks. The mull was sandwiched between sodium chloride plates before being placed in the spectrometer. Analysis was performed on a Nicolet Avantar 320 FT-IR with EZ OMNIC version 6.0a software. Measurements were performed again at room temperature (21OC). The sample was scanned 32 times with wavenumbers range 4000-500cm-1. The background spectrum was again subtracted from the sample spectra.

40

4.3 Results and Discussion

Nujol Mull Nujol mull preparation is a standard mode of FT-IR analysis. It is relatively easy to prepare and can provide a protective coating to reactive samples during acquisition of the spectra. However, it is highly important to prevent the saturation of the sample with the oil and this leads to erroneous spectra as the Nujol bands will dominate, silencing the sample's peaks. This was of particular concern during the silk analysis as the amount of sample available was limited. Several mulls were prepared, each with an increased amount of silk, until the sample bands became visible.

Figure 16. 16A. FT-IR spectrum of Nujol oil, 16B. FT-IR spectrum of Tegenaria silk.

41

As can be seen from figure 16B above, there are three distinctive peaks that are produced by the silk and were not present in the Nujol oil spectrum.

Table 13. IR bands from silk Location of peak (cm-1) 3296.38 1654.82 1517.10 Associated structure/Functional group N-H stretch -helix and random coil -helix

The peak at 3296.38 cm-1 at is characteristic of an N-H stretch of a secondary amine. The band is relatively weak and sharper than an alcohol O-H stretch which occurs in the same region. Secondary amines are produced after the condensation reaction of two amino acids to form a peptide bond in proteins, such as spider silk. The peaks at 1654.82 cm-1 and 1517.10 cm-1 are characteristic of -helical structures and random coil of the silk protein molecule. Amide I bands are found between 1652-1660cm-1 and are from stretching vibrations of the C=O of the amide. These correspond to helical structures such as -helix. The protein in this silk is not in -sheet conformation as infrared bands between 16441648cm-1 would have been detected. The presence of an additional peak at 1695 4cm-1 would correspond to anti-parallel -sheets. Furthermore, if the protein had solely a random coil secondary structure then a broad band would have been observed between 1644-1648cm1

, thus indicating the combination of random coil and -helix conformations.

ATR FT-IR spectroscopy

An ATR FT-IR spectrum of the Tegenaria silk was also produced. The table below shows the dominant peaks observed.

42

98.0 96 94 92 90 88 86 84 82 80 78 76 74 72 %T 70 68 66 64 62 60 58 56 54 52 50 48 46 45.0 4000.0 3600 3200 2800 2400 2000 1800 cm-1 1600 1400 1200 1000 800 650.0
1643.09 1643.09 1515.99 1068.80 1068.8 1235.37 1164.89 1164.8 2964.38

2964.38

3285.47

3285.47

1454.52

1454.52

1235.37

1515.99

Figure 17. ATR FT-IR spectrum of the Tegenaria silk

Table 14. Main IR bands observed. Location of peak (cm-1) 3285.47 2964.38 1643.09 1515.99 1454.52 1235.37 1164.80 1068.80 Associated structure/Functional group N-H stretch -CH- asymmetric stretching -helix and random coil -helix -CH (CH3) bending -C-O- stretch -C-O- stretch -C-O- stretch

43

As there was no sample preparation for this analysis, all the peaks correspond to the silk sample. Table 14 above shows the IR bands that were observed and the structural element they are characteristic of. The three bands that were detected in the Nujol mull sample, listed in table 13, were also present in this sample. They once again showed random coil and helix conformations in the silk protein. The other bands that were detected corresponded to CH- bending and stretching and a -C-O- stretch. These are due to the silk protein and would be found in the FT-IR spectrum of any protein. In the study by Van Nimmen et al (2008)(36), it was stated that ATR FT-IR spectroscopy can sometimes result in slight shifts of strongly absorbing bands to lower frequencies and distortion of peak shape. This is a result of the refractive index suddenly changing in the region of an absorption which can cause loss of the criterion of internal reflection. In comparison with the Nujol sample, a shift of bands to a lower frequency was not observed. The bands that were common to both samples were at a slightly higher frequency in the ATR FT-IR spectra but the difference was negligible. With regards to a distortion in peak shape, a slight distortion could be seen at the beginning of some peaks to a small degree but most produced Gaussian shaped peaks and the software was easily able to detect the point of the peak.

ATR-FTIR was seen to be a significantly better technique than Nujol preparation for silk analysis. There was no sample preparation at all required for ATR FT-IR analysis. This made it more time efficient and also allowed for the sample to be re-used for other analyses, as it was not a destructive technique. It also required less sample which is a huge benefit in silk analysis as obtaining silk is a very slow process, making it extremely valuable. Additionally it allowed generation of a complete spectrum of just the sample which permitted easier interpretation.

Spectra produced by both techniques are available for viewing in the appendix of this report.

44

Chapter 5. Conclusions and Further work

5.1 Conclusions
The aim of this report was to document the chemical composition of spider silk, with additional focus on the lipids present, as well as to report the findings of the analysis performed on Tegenaria silk.

It was seen that spider silk is currently under intensive study due to its impressive mechanical properties such as high tensile strength and elasticity. These properties are very attractive for commercial applications and have prompted a recent surge in investigations into the chemical composition and structure of spider silk. Whilst a lot is known about the primary component of the silk, complex protein molecules, the lipid content hasnt been investigated as thoroughly. The lipids comprise 3-5% of the fibres and are found in a coat on the outside of the silk strands. The function of this layer is also still unknown.

GCMS analysis performed on the silk of a Tegenaria spider revealed the presence of numerous lipid classes. These included fatty acids, straight chain n-alkanes, sugars, sterols and n-alkanols. The fatty acids were determined to be the most abundant compound. A complete characterization of the lipid content of the silk and the relative abundances of the components were obtained.

The series of images generated by SEM showed a variety of silk structures in Tegenaria silk and the presence of different types of silk. Rounded bilateral strands as well as strands containing glue droplets were observed which are likely to correspond to major ampullate silk and flagelliform silk respectively. Ridged strands, semi-braided fibres, multiple stranded fibres and minor ampullate silk were also observed. Furthermore, a variation in silk diameter was noted and possible functions assigned to each of the strands.

EDX analysis was performed too on Tegenaria silk. Carbon, oxygen and nitrogen were present on both fresh and extracted silk. This indicated that there is likely a source on nitrogen on the surface of the lipids that was detectable. It is possible that it originates from the glue droplets which were viewed previously under SEM. 45

Finally, two modes of IR analysis were used to investigate the secondary structure of the Tegenaria silk. Nujol mull and ATR FT-IR spectroscopy both revealed bands in the Amide I and Amide II region which corresponded to random coil and -helix conformations in the silk protein. ATR FT-IR was also determined to be the better technique for silk analysis.

5.2 Further work

In terms of silk analysis, there is plenty of room for further research. As it is a relatively new area of study a great amount remains unknown. At present there has not yet been a full protein sequence determined, the lipid content has only been fully characterized for one spider species, out of 37,000, and the function of the lipid layer remains unknown. The presence of low molecular mass compounds (LMMs) has also been reported on silk. These compounds have not yet been characterized either or a function assigned. These are only a few of many possible options for study.

Specific areas for further work, related to this project, would be to further investigate the use of SPE as a technique for fractionating lipid samples. As previously stated this technique is potentially very beneficial for improving chromatograms and making identification easier. For this to be investigated a sufficient amount of silk would need to be acquired as it is likely that an insufficient quantity of sample hindered the process during the project.

In relation to SEM/EDX analysis it would be interesting to cryomicrotome silk bundles so the cross-section of silk strands could be studied. The elemental composition of the strands could also be mapped using EDX to ascertain if there is a detectable difference between the lipid coat and the protein core. Additionally the glue droplets could be studied using EDX.

46

References
1. Vollrath, F. (2000) Strength and Structure of Spiders Silk. Reviews in Molecular Biotechnology, 74(2), pp.67-83. 2. Romer, L. and Scheibel, T. (2008) The Elaborate Structure of Spider Silk Structure and Function of a Natural High Performance Fiber. Prion, 2(4), pp.154-161. 3. Vollrath, F. (2006) Spider silk as a model biomaterial. Applied Physics A, 82(2), pp.205-212. 4. Altman, G. H., Diaz, F., Jakuba, C., Calabro, T., Horan, R. L., Chen, J., Lu, H., Richmond, J. and Kaplan, D. L. (2003) Silk-based biomaterials. Biomaterials, 24(3), pp. 401-416. 5. Schulz, S. (1997) The Chemistry of Spider Toxins and Spider Silk. Angewandte Chemie-International Edition in English, 36(4), pp.315-326. 6. http://australianmuseum.net.au/Spider-silk-production, accessed 16/12/10. 7. Hardy, J. D. and Scheibel, T. R. (2010) Composite Materials based on Silk Proteins. Progress in Polymer Science, 35(9), pp.1093-1115. 8. Gosline, J. M., Guerette, P.A., Ortlepp, C. S. and Savage K. N. (1999) The Mechanical Design of Spider Silks: From Fibroin Sequence to Mechanical Function. Journal of Experimental Biology, 202(23), pp.3295-3303. 9. Sponner, A., Vater, W., Monajembashi, S., Unger, E., Grosse, F. and Weisshart, K. (2007) Composition and Hierarchical Organisation of a Spider Silk. Plos One, 2(10), e998 pp.1-8. 10. Du, N., Liu, X. Y., Narayanan, J., Li, L., Lek Min Lim, M. and Li, D. (2006) Design of Superior Spider Silk: From Nanostructure to Mechanical Properties. Biophysical Journal, 91(12) pp.4528-4535. 11. Hu, X., Vasanthavada, K., Kohler, K. McNary, S., Moore, A. F. M., and Vierra, C. A. (2006) Molecular Mechanisms of Spider Silk. Cellular and Molecular Life Sciences, 63(17), pp.1986-1999. 12. Saravanan, D (2006) Spider Silk- Structure, Properties and Spinning. Journal of Textile and Apparel, Technology and Management, 5(1), pp.1-20. 13. Zhang, Y., Dai, L. and Ou-Yang, Z. C. (2004) Amino Acid Composition- Dependent Elasticity of Spider Silk. International Journal of Modern Physics B, 18(17-19), pp.2516-2522. 47

14. Vollrath, F. and Knight, D. P. (2001) Liquid Crystalline Spinning of Spider Silk. Nature, 410(6828), pp.541-548. 15. Zhang, M., Yi, T., Zhang, Y., Zhang, L., Wu, W., Zhang, A. and Pan, Z. (2011) Ornithoctonus huwenna spider silk protein attenuating diameter and enhancing strength of the electrospun PLLA fiber. Polymers for Advanced Technologies, 22(1), pp.151-157. 16. Yang, L., Hedhammar, M., Blom, T., Leifer, K., Johanson, J., Habibovic, P. and Van Blitterswijk, C. A. (2010) Biomimetic calcium phosphate coatings on recombinant spider silk fibres. Biomedical Materials, 5(4), Sp. Iss. SI, article number 045002. 17. Swanson, B. O., Anderson, S. P., DiGiovine, C., Ross, R. N. and Dorsey, J. P. (2009) The evolution of a complex biomaterial: The case of spider silk. Integrative and Comparative Biology, 49(1), pp. 21-31. 18. Vollrath, F. (1999) Biology of spider silk. International Journal of Biological Macromolecules, 24(2-3), pp.81-88. 19. Creager, M. S., Jenkins, J. E., Thagard-Yeaman, L. A. (2010) Solid-State NMR Comparison of Various Spiders' Dragline Silk Fiber. Biomacromolecules, 11(8), pp.2039-2043. 20. Vollrath, F. (2006) Spider silk: Thousands of nano-filaments and dollops of sticky glue. Current Biology, 16(21), pp.925-927. 21. Bielecki, K. (2009) Spider silk structures as seen with a scanning electron microscope. Bios, 80(4), pp.170-175. 22. Knight, D. P. and Vollrath, F. (2001) Changes in element composition along the spinning duct in a Nephila spider. Naturwissenschaften, 88(4), pp.179-182. 23. Kaplan, D., Adams, W. W., Farmer, B. and Viney, C. (1994) Silk Polymers: Materials Science and Biotechnology. Washington: American Chemical Society. 24. Hu, X. Y., Yuan J, Wang, X. D., Vasanthavada, K., Falick, A. M., Jones, P. R., La Mattina, C and Vierra, C. A. (2007) Analysis of Aqueous Glue Coating Proteins on the Silk Fibers of the Cob Weaver, Latrodectus Hesperus. Biochemistry, 46(11), pp.3294-3303. 25. Van Beek, J. D., Hess, S. Vollrath, F. and Meier, B. H. (2002) The molecular Structure of Spider Dragline Silk: Folding and Orientation of the Protein Backbone. Proceedings of the National Academy of Sciences of the United Stated of America, 99(16), pp.10266-10271.

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26. Sherman, P. M. (1994) The Orb-Web- An Energetic and Behavioral Estimator of a Spiders Dynamic Foraging and Reproductive Strategies. Animal Behaviour, 48(1), pp.19-34. 27. Diederichsen, U., Lindhorst, T. K., Westermann, B. and Wessjohann, L. A. (1999) Bioorganic Chemistry: Highlights and New Aspects. Germany: Wiley-VCH 28. Schulz, S. (2001) Composition of the Silk Lipids of the Spider Nephila clavipes. Lipids, 36(6), pp.637-647. 29. Schulz, S. (1997). Mass spectrometric determination of methyl group positions in long chain methyl ethers and alcohols via nitriles. Chemical Communications, 1(10), pp.969-970. 30. Suter, R. B., Shane, C. M. and Hirscheimer, A. J.(1987) Communication by Cuticular Pheromones in a Linyphiid Spider. Journal of Arachnology, 15(2), pp.157-162. 31. Schulz, S. and Toft, S. (1993) Branched long chain alkyl methyl ethers: A new class of lipids from spider silk. Tetrahedron, 49(31), pp.6805-6820. 32. http://wiki.britishspiders.org.uk/index.php5?title=Tegenaria_gigantea_saeva, accessed 17/05/11. 33. Rousseau, M. E., Hernandez, D., West, M., Hitchcock, A. P. and Pezolet, M. (2007) Nephila clavipes spider dragline silk microstructure studied by scanning transmission x-ray microcopy. Journal of the American Chemical Society, 129(13), pp.3897-3905. 34. Hakimi, O., Knight, D. P., Knight, M. M., Grahn, M. F. and Vadgama, P. (2006) Ultrastructure of Insect and Spider Cocoon Silks. Biomacromolecules, 7(10), pp.2901-2908. 35. Sensenig, A., Agnarsson, I and Blackledge, T. A. (2010) Behavioural and Biomaterial Coevolution in Spider Orb Webs. Journal of Evolutionary Biology, 23(9), pp.18391856. 36. Van Nimmem, E., De Clerck, K., Verschuren, J., Gellynck, K., Gheysens, T., Mertens, J. and Van Langenhove, L. (2008) FT-IR spectroscopy of spider and silkworm silks: Part 1. Different sampling techniques. Vibrational Spectroscopy, 46(1), pp.63-68. 37. Ene, R., Papadopoulos, P. And Kremer, F. (2010) Partial deuteration probing structural changes in supercontracted spider silk. Polymer, 51(21), pp. 4784-4789. 38. Agnarsson, L., Boutry, C., Wong, S. C., Baji, A., Dhinojwala, A., Sensenig, A. T. and Blackledge, T. A. (2009) Supercontraction forces in spider dragline silk depend on hydration rate. Zoology, 112(5), pp.325-331. 49

Appendix

50

Pooled sample, Control, Tegenaria sample

51

SPE samples: Neutral lipid fraction, PHA fraction and Polar lipids fraction.

52

Spider Silk Analysis

26/04/2011 16:43:20

Project Notes: Non extracted silk- contains lipid coat (C,H,O) Extracted silk- no lipid coat-protein layer (C,H,O,N)

Project: Spider Silk Analysis Owner: Inca Operator Site: Site of Interest 3

Sample: Sample 1 Type: Default ID: Non extracted silk

Spectrum processing : Peaks possibly omitted : 1.656, 2.134 keV Processing option : All elements analyzed (Normalised) Number of iterations = 3 Standard : C CaCO3 1-Jun-1999 12:00 AM N Not defined 1-Jun-1999 12:00 AM O SiO2 1-Jun-1999 12:00 AM Element CK NK OK Totals App Conc. 348.45 20.67 168.53 Intensity Corrn. 2.1289 0.6629 2.0924 Weight% 59.43 11.32 29.25 100.00 Weight% Sigma 0.95 1.10 0.75 Atomic% 65.24 10.66 24.10

Sample Notes: Non extracted silk strands

Spectrum Label: Spectrum 1 Livetime 300.0 s Acquisition geometry ( degrees ): Tilt = 0.0 Azimuth = 0.0 Elevation = 35.0 Accelerating voltage = 3.50 kV Total spectrum counts = 206364 Sample data : Strobe : Energy (eV) 3.8 Resn. (eV) 40.84 Area 2909677

Comment:

53
Signature_____________________

Spider Silk Extracted Silk Analysis

26/04/2011 17:21:32 17:04:59

Project Notes: Sample 1- non extracted silk- contains lipid coat (C,H,O) Sample 2- extracted silk- no lipid coatprotein (C,H,O,N) Project: Extracted Spider Silk Silk Analysis Owner: Inca Operator Site: Site of Interest 1 4

Sample: Sample 1 Type: Default ID: Non extracted silk

Spectrum processing : Peaks possibly omitted : 1.651, 1.044, 2.132 1.661,keV 2.132 keV Processing option : All elements analyzed (Normalised) Number of iterations = 3 2

Sample Notes:

Standard : C CaCO3 1-Jun-1999 12:00 AM N Not defined 1-Jun-1999 12:00 AM O SiO2 1-Jun-1999 12:00 AM Element CK NK OK Totals App Conc. 230.14 369.53 -1.56 28.71 42.24 180.37 Intensity Corrn. 2.1453 2.1290 0.5706 0.6719 2.0170 2.0818 Weight% 57.29 85.50 -2.18 14.11 16.69 28.60 100.00 Weight% Sigma 2.36 0.90 2.40 1.05 1.27 0.73 Atomic% 63.06 88.92 -1.95 13.31 13.03 23.63

Spectrum Label: Spectrum 1 Livetime 300.0 s Acquisition geometry ( degrees ): Tilt = 0.0 Azimuth = 0.0 Elevation = 35.0 Accelerating voltage = 3.50 kV Total spectrum counts = 205752 244038 Sample data : Strobe : Energy (eV) 3.8 Resn. (eV) 40.87 40.84 Area 2907889 2910903

Comment:

54
Signature_____________________ __________

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56

57

58

ATR FT-IR
98.0 96 94 92 90 88 86 84
1164.89 2964.38

82 80 78
3285.47 1454.52 1235.37 1068.80

76 74 72 %T 70 68 66 64 62 60 58 56 54 52 50 48 46 45.0 4000.0 3600 3200 2800 2400 2000 1800 cm-1 1600 1400
1643.09 1515.99

1200

1000

800

650.0

59

98.0 96 94 92 90 88 86 84 82 80 78 76 74 72 %T 70 68 66 64 62 60 58
1516.01 3285.44 1454.59 1235.62 1069.12 2964.36 1165.76 700.00

56 54 52 50 48 46 45.0 4000.0 3600 3200 2800 2400 2000 1800 cm-1 1600 1400 1200 1000 800 650.0
1643.32

60