Bioanalytical strategies for the quantification of xenobiotics in biological fluids and tissues 7

Chapter Chapter Detection of endorepellin in ovarian tumor serum and plasma samples by atomic force microscopic imaging study: Insights to early detection of ovarian tumor 133 Chapter 7.1 Preface to Chapter Recent advances in angiogenesis research and vascular biology have led to the discovery of a powerful angiogenesis protein inhibitor named endorepellin. Endorepellin is found to be in higher concentration in normal healthy humans and lower concentration in cancer patients hence it has been identified as a potential cancer biomarker and therapeutic drug. For the first time, an attempt was made to apply the atomic force microscopic study on tumor and control serum samples to compare the levels of endorepellin expression in both tumor and control samples. To identify and understand the biological activities and chemistry involved, computational modelling of the protein was done. Conventional proteomics were done in this study in a bid to differentiate endorepellin expression in the tumour and control samples. Plasma (n=2) and serum (n=2) from healthy human and cancer patient were used. Through the Bradford assay, it was apparent that the total protein concentration for both healthy and cancerous samples was similar and fall within a range of 69 mg mL-1 to 75 mg mL-1. Protein profiling was done using one-dimensional polyacrylamide gel electrophoresis (1D SDS-PAGE) and LG3 was successfully found to be less expressed in cancerous plasma and serum than in healthy samples. That was further proved by AFM imaging study on tumor and control serum and plasma samples. 134 Chapter 7.1 Introduction Angiogenesis is the development of new blood capillaries and is widely involved in several physiologic and pathologic processes such as invasive tumour growth [1]. In particular, it was first hypothesized by Folkman that tumour-growth is angiogenesis dependant in 1971 [2]. However, it was only after the discovery of the first angiogenesis inhibitor and the purification of the first angiogenesis protein in the mid 1980s that resulted in the widespread acceptance of the concept. The discovery of an efficacious angiogenesis protein inhibitor named endorepellin in recent times; signify an exciting potential breakthrough in the detection of tumour cancers and its subsequent cancer therapies [3]. Endorepellin (85-kDa) is the C-terminal domain of a large modular protein called Perlecan (~470kDa) which is composing of five structural domains. Perlecan is a basement membrane heparan sulphate proteoglycan that is involved extensively in vascular growth and tumour angiogenesis. Endorepellin consists of three laminin-like globular domains (LG1-LG3) and is found to interact solely with the α2β1 integrin, a receptor for collagen I, in platelets and endothelial cells. Being one of the key receptors of endothelial cells, α2β1 provides vital support for vascular endothelial growth factor (VEGF) signalling, endothelial cell migration, and tumour angiogenesis [4]. Therefore, by binding to α2β1 integrin, endorepellin causes disorder to the cell’s cycloskeleton and adhesion properties [5]. On the whole, endorepellin was shown to inhibit three major steps in angiogenesis namely adhesion, migration and morphogenesis Research studies had shown that systematic delivery of human recombinant endorepellin to tumour xenograft mice causes a considerable suppression of tumour growth and metabolic rate as brought about by a continuous 135 Chapter down-regulation of the tumour angiogenic network [6]. Apart from down-regulating pro-angiogenic proteins, endorepellin can also attach to endostatin (another matrixderived inhibitor of angiogenesis that had been tested in clinical trials) and work against its anti-angiogenic effects [3]. The superior anti-angiogenic abilities of endorepellin further strengthen the belief that it will serve as a better biomarker and therapeutic drug. In particular, the last laminin-like globular domain, LG3 (~26kDa) is found to acquire most of the biological activities and thus has most of the anti-angiogenesis ability. LG3 can interact and be released by partial proteolysis during physiologic and pathologic processes such as tissue remodelling and cancer growth [7]. This is proven by the fact that LG3 fragments were found in the urine of patients with end-stage renal failure and chronic allograft nephropathy, and in the amniotic fluid of pregnant woman [8-10]. More importantly, it was shown for the first time that circulating LG3 levels in human breast cancer plasma was significantly lower than the LG3 levels in healthy human plasma, indicating endorepellin, more specifically, LG3 as a potential biomarker for cancer detection, progression and invasion [11]. Apart from its anti-angeogenis activity and its ability to reduce tumour to a manageable size or inhibit tumour growth, other intrinsic characteristics of endorepellin also justify its selection for this study. Being a protein-based inhibitor, it does not induce resistance and the toxicity is low. It is able to work in low concentration (i.e. nM) and it may also exert an anti-adhesive action on certain tumour cells. It shows better anti-angiogenic properties and therefore a greater potential as a tumour biomarker and therapeutic drug. Endorepellin is found to be in higher concentration in normal healthy humans and lower concentration in cancer patients. 136 Chapter The malignant transformation of a normal epithelial cell is generally thought to be caused by genetic alterations or mutations that disrupt the regulation of proliferation and apoptosis, in turn leading to an altered protein expression and modification [12]. Alterations in protein levels can be detected not only in the cancer cells, but also in the blood and other body fluids into which these proteins are secreted. This can therefore aid in the identification of a normal cell transforming into a cancerous state. Hence analysis of those body fluids by proteomic studies for quantification of endorepellin will lead to the clue about the state of the tumour cells. Electrophoresis is the separation of macromolecules in an electrically charged field. For this, a support medium such as polyacrylamide or agarose is required. Gel electrophoresis is a simple way to separate proteins prior to downstream detection or analysis. PAGE is most commonly used to separate proteins in a sample based on their molecular weight (or length of polypeptide chain). However, the general electrophoresis methods cannot be used to separate proteins according to molecular weight alone because the mobility of a substance in the gel is influenced by both charge and size. In order to overcome this, the proteins undergoing electrophoresis are treated with SDS, an anionic detergent, so that proteins have a uniform charge. The method of SDS-PAGE that is currently being used, involving the use of a Tris-glycine running buffer to carry out electrophoresis, was first described by Laemmli and is better known as the Laemmli method [13]. Atomic force microscopy is an imaging technique which permits the investigation of molecules in their native physiological buffer condition without subjecting the sample to harsh treatments such as drying, crystallizing or vaporizing in vacuum, thereby not limiting the range of measurable dynamical properties of the 137 Chapter sample. This feature made this technique highly suitable for topographical imaging of biological samples [14]. AFM can provide nanometer-resolution images of living cells in gaseous and liquid environments. In an AFM, a sharp stylus (approximately tenths of a nanometer) attached to the end of a cantilever is approached to the surface. As a consequence, a force appears between the tip and surface that can be attractive or repulsive causing the cantilever to bend. When this bending is controlled with a feedback algorithm, it is possible to obtain a topographic map by scanning the surface in a plane perpendicular to the tip. By using this technique individual protein molecules in aqueous solutions can be imaged directly at sub molecular resolution. If suitable antibody reagents were available, this technology could be used to detect the presence of a specific protein by identifying its protein-antibody complex. In this present study, the structure and environment of the LG3 domain in the endorepellin was identified using homology modelling and the presence of endorepellin in plasma and serum samples was established by conducting conventional proteomic studies i.e. SDS-PAGE for the quantification, separation and identification of LG3 domain. Further, the expression of endorepellin in tumour plasma and serum samples was detected by Atomic force microscopic imaging studies and compared with control plasma and serum samples. 7.2 Experimental 7.2.1 Chemicals and reagents Acetic acid, Acetone, Formic acid, Tris base (Merck); Bovine serum albumin (BSA) standards, Bradford dye, bromophenol blue, dithiothreitol (DTT), glycerol, glycine, (Sigma-Aldrich); ammonium persulphate, 30% bis/Acrylamide, Precision 138 Chapter Plus protein all blue standards, SDS, N,N,N',N'-Tetramethylethylenediamine (TEMED) (Bio-Rad Laboratories, Hercules, CA, USA); Silver Stain plus kit (Bio-Rad laboratories), mouse monoclonal [a74] to heparan sulfate proteoglycan antibody (Anti-HSPG2) (Abcam, Cambridge, UK), 7.2.2 Water and solutions Autoclaved water, 1x phosphate-buffered saline (PBS); 5x SDS/Glycine electrophoresis buffer (15.1 g tris base, 72 g glycine and g SDS); Silver stain fixative solution (40% methanol, 10% acetic acid (v/v); Silver stain stop solution (5% acetic acid, 95% water); 2x loading buffer (0.313 M Tris-HCl pH 6.8 at 25oC, 10% SDS, 0.05% bromophenol blue, 50% glycerol, and 0.5 M DTT); upper Tris solution (0.5 M Tris [pH 6.8], 0.4% SDS); lower Tris solution (1.6 M Tris [pH8.8], 0.4% SDS); rehydration buffer (7 M urea, M thiourea, 100 mM DTT, 4% CHAPS, 0.5% carrier ampholytes pH 4–7, 0.01% Bromophenol blue (BPB) and 40 mM Tris). 7.2.3 Hardware and equipment P-2, P-10, P-20, P-100, P-200 and P-1000 pipettes (eppendorf); 96 well microtiter plate (Tecan Asia); 0.75 mm spacer plates, short glass plates, gel casting stand and combs (Bio-Rad Laboratories); GS-800 calibrated densitometer, UltraRocker Rocking Platform (Bio-Rad Laboratories); Pχ2 programmable thermal cycler (Thermo Hybraid, Middlesex, TW, USA); bench top microcentrifuge for 0.5and 1.5 ml polypropylene tubes (Sanyo Gallenkamp PLC, Loughborough, UK); PowerWaveX Select Microplate Spectrophotometer (BioTek, Winooski, VT, USA); pH meter, Weighing Balance (Sartorius). 139 Chapter 7.2.4 Computer software The PyMOL molecular graphics system (DeLano Scientific, Palo Alto, CA, USA), Accerlys Discovery Studio 2.0 client (Accerlys Inc, San Diego, CA, USA), KC4 (BioTek, Winooski, VT, USA); PDQuest version 7.2 software package (Bio-Rad Laboratories,) GwyddionTM 2.29 (Czech republic). 7.2.5 Atomic force microscopy The imaging of endorepellin expression was performed using NanoMan AFM system (Veeco metrology group, USA) which allows contact and tap mode image, multichannel data acquisition, and operates under ambient laboratory conditions, in vacuum, or in solution. The system equipped with calibrated silicon nitride AFM cantilever (OTR8- 35) with force constant of 0.57 N/m, tip size of 15 nm and resonant frequency 300 kHz (Veeco). 7.2.6 Preparation of plasma and serum samples Plasma (n=2) and serum (n=2) samples from a healthy being and ovarian cancer patient were each obtained from the Department of Obstetrics & Gynaecology, National University Hospital, Singapore. The fluids were centrifuged at 15,000 rpm for 10 at 4˚C. The supernatants were then divided into aliquots of mL, snap frozen in liquid nitrogen, and stored at -80˚C until analysis. 7.3 Methodology 7.3.1 Homology modelling Homology Modelling is fundamentally made up of two principles. Firstly, the structure of a protein is distinctly identified by its amino acid sequence [15]. This implies that the sequence information alone is sufficient to obtain the protein structure. Secondly, the structure is more highly conserved than the sequence, 140 Chapter suggesting that the structure is more stable and changes less significantly during evolution [16]. As such, similar sequences are assumed, and later proven, to fold into practically identical structures and that distantly related sequences will still adopt similar structures [17]. It is with these principles that allow modelling of an unknown target based on the sequence similarity with other homologous proteins that have known crystal structures. These homologous proteins are referred as templates in this chapter. The search for templates was first carried out using the PSI-BLAST and BLASTp server at NCBI. Unfortunately, the search did not return with substantial results, thus another technique called the fold recognition was also employed. This technique specifically searches for similar secondary structures (such as the folding of alpha helix or beta sheets etc) in addition to searching for similar sequences. Through LOMETS, an automatic mail server for protein secondary structure prediction, templates with the highest identities match were obtained (Table 7.1). The templates were named as according to their Protein Data Bank (PDB) number. Table 7.1 Top templates results obtained through secondary structure prediction Template 1dyk Title Source Resolution Similarity Identity House mouse 2.00 Å 42.90% 25.00% Chicken 1.42 Å 41.60% 21.50% - 1.68 Å 42.20% 22.60% Laminin alpha chain LG 4-5 domain Modulation of agrin 1pz7 function by alternative splicing and Ca2+ binding 1dyk + 1pz7 - 141 Chapter From Table 7.1, it is evident that murine laminin α2LG4-5 domain has the highest percentage of identity (25%) when matched with the endorepellin LG3 sequence. The low similarity and identity of all the templates are typical of homology modelling among LG domains [18]. However, the low resolution of 1dyk posed as an obstacle to creating a good homology model. With respect to this, the templates 1dyk and 1pz7 were superimposed onto each other in an attempt to resolve the resolution problem while not comprising much on the identity percentages. The newly generated multiple sequence alignment was then aligned with the target LG3 sequence (Figure 7.1), thereby creating the basis of the final LG3 homology model. The overall sequence identity obtained is 22.6% with the model having a resolution of 1.68Å. 7.3.2 Protein quantification, sample preparation, separation and identification of LG3 7.3.2.1 Total protein quantification by the Bradford assay The Bradford method is a colorimetric assay technique used to determine protein concentration in a sample. It uses the Coomassie Brilliant Blue G-250 dye, which has a maximum absorbance at 595 nm when bound to proteins. The dye binds primarily to lysine and arginine residues on the protein, where it becomes ionised and its maximum absorbance increases. The increase in absorbance at 595 nm is thus proportional to the amount of protein present. As the Bradford assay is only linear over a short range between 100 and 1500 μg mL-1, samples were diluted with a 100 factor before quantification begins. Therefore, µl of each samples were added with 495 µl of 1x PBS. Next, 250 µl of the Bradford (Coomassie Brilliant Blue G-250) dye were added to µl of each standards and samples in a 96-well microtiter plate. Duplicate standards and samples 142 Chapter The first step was to immobilize the antibody on the substrate. The stock solution of anti-HSPG2 was diluted and fold into Milli Q water. Twenty microliters of the Anti-HSPG2 was drawn off with a pipette and deposited on the freshly cleaved mica substrate. Then the antibody covered mica substrate was incubated for approximately 20 sec before rinsing in excess Milli Q water to remove any weakly adsorbed antibody and residual salt deposits. After washing, the substrate was dried in a stream of dry N2 gas (1 bar pressure at a distance of several cm). Rinsing can be achieved by running up to ml of Milli Q water across the mica sample while it is tilted at a 30 to 45° angle. The AFM imaging was done immediately after evaporation of the solvent to prevent contaminations. Subsequently, the target protein in the sample was applied to the antibody adsorbed mica substrate and allowed to dry under a flow of N2 gas. Immediately after solvent evaporation, the antigen-antibody layer washed three times with water to remove any residual deposits or loosely adsorbed proteins. The samples were then allowed to dry completely and then incubated in a humid chamber at room temperature for a specified period (60 min, 30 min) of time. The amount of antibody binding depends on several factors, including degree of washing with water, concentration of antibody, and other incubation conditions. The conditions of binding with surface adsorbed protein are summarized in Table 7.3. In the control experiments, Anti-HSPG2 was used with the same incubation times and at the same concentrations as of tumor samples. 147 Chapter Table 7.3 Summary of the Reaction Conditions for tumor plasma, serum, control samples Sample size 20 µl Anti-HSPG2 Conc, µg/mg 0.5 0.25 0.5 0.25 7.4 Results and discussion 7.4.1 Homology Modelling incubation time, h 0.5 0.5 0.5 1 Being a novel inhibitor of angiogenesis, endorepellin was coined as a breakthrough in angiogenesis research with its promising capacity as an exceptional biomarker and cancer therapeutic drug[20]. However, as of today, there is no known crystal structure of the protein available. Therefore, in an attempt to further comprehend the molecular factors of the biological activity and the chemistry involved, computer aided structure model of endorepellin (or more specifically LG3) was constructed using the 26kDa LG3 fragment sequence (Figure 7.1). Figure 7.1 Part of human heparan sulphate proteoglycan (HSPG2) sequence, depicting endorepellin (D3681-S4381) and LG3 (in red G4182-S4381). 148 Chapter Homology modelling methodology was used to construct structure model of endorephilin, which is based on availability of homologous experimental crystal structure (template) and proximity of their sequences identities. Two templates (PDB ID: 1pz7 and 1dyk) were obtained from the database search and these were found to be having low resolution. To circumvent the problems associated with individual templates, multi-template paradigm was used (details in methodology section). The sequence alignment between target (human LG3) and templates is shown in the figure 7.2. The multiple sequence alignment (figure 7.2) in conjunction with experimental structures was used to generate structural model of human LG3 protein. 5 12 13 10 11 14 15 Figure 7.2 Sequence alignments of 1pz7, 1dyk and LG3. Identical alignment of residues and similar alignment of residues are depicted by dark blue and light blue colouring respectively. Red boxes depict residues that are found to coordinate to calcium. Thus obtained model of LG3 was thoroughly optimized and validated (details in methodology). The model consists of β-sheets or β-sandwich made up of 15 antiparallel β strands which are depicted as the purple numerical arrows in Figure 7.2. Most of these predicted β strands also coincides with areas of high identity/homology between the LG3 sequence and the combined template. More importantly, LG3 was found to be calcium coordinated by residues namely – Aspartic acid4258, Leucine4275, Asparagine4323, Alanine4325 and Asparagine4327 (Figure7.3 (A)) and 149 Chapter this coordination area is predicted to be the receptor for its biological activities since calcium ion was also found to be partially exposed. The chemical structures of the coordinating residues are shown in Figure 7.3 (B). (A) (B) Figure 7.3 (A) A ribbon diagram of the LG3 model showing the 15 anti-parallel β strands (depicted as arrows) and the calcium ion (depicted as the pink sphere). (B) Diagram showing the calcium ion (green sphere) with coordinating residues. The values on the dotted yellow lines depict the distance between the species in angstroms. 7.4.2 Proteomic study 7.4.2.1 Total protein quantification in samples using the bradford assay The total protein concentration in each of the plasma and serum samples to be used in this study must be known so that in the subsequent stage of protein separation by SDS-PAGE, the same amount of protein for each plasma and serum sample is loaded. This is to ensure that the protein band/spot intensity (which reflects the amount of that protein present) between different samples will be without bias and can be compared. The total protein concentration in each of the plasma and serum samples is shown in Table 7.4 and a sample absorbance versus concentration plot is shown in Figure 7.4. 150 Chapter Table 7.4 Total protein concentration in samples as measured using the Bradford assay Sample Type Control plasma Total protein concentration (mg/ml) Sample Sample 75.81 74.075 Average (mg/ml) 74.943 Tumor plasma 73.538 74.685 74.112 Control serum 71.157 71.98 71.569 Tumor serum 69.048 72.723 70.886 Figure 7.4 Sample plot of absorbance vs concentration (mg mL-1) for plasma and serum samples. From Table 7.4, it can be observed that the total protein concentration in both healthy and cancerous samples was similar. In addition, the protein concentration level in serum samples was found to be lower than the concentration level in plasma samples. This could be explained by the fact that clotting proteins such as fibrinogen had been removed in serum samples. Generally, this result is expected, as the concentration differences in healthy and cancerous samples will only be more prominent when the focus is on the individual biomarker’s concentration and not the total protein concentration. 151 Chapter 7.4.2.2 SDS-PAGE and gel staining SDS has a very hydrophobic end (the lipid-like dodecyl part) and a highly charged sulphate group. It interacts with hydrophobic amino acids in proteins and disrupts the secondary and disulphide bond-linked tertiary structures of the proteins, which depends largely on interactions between hydrophobic amino acids in their core. Being anionic, SDS has a negative charge over a wide pH range. Therefore, mixing the proteins in a biological sample with SDS will result in proteins having the form of linear polypeptide chains coated with negatively charged SDS molecules. The polyacrylamide gel is a cross-linked matrix that acts as a threedimensional mesh. As the negatively charged protein molecules in the samples are drawn towards the positively charged anode, they encounter resistance provided by the polyacrylamide gel, thereby restraining larger molecules from migrating as fast as the smaller molecules. As the mass to charge ratio is nearly the same for all the SDSdenatured polypeptides, the final separation of proteins is dependent almost entirely on the differences in relative molecular mass of polypeptides. The speed of migration is dependent on the size of the ‘pores’ in the gel mesh which is in turn determined by the percentage of acrylamide present in the gel. The polyacrylamide gel consists of parts: the stacking gel and the resolving gel. The stacking gel is used to form the wells in which the protein sample is loaded. It has a very low acrylamide concentration, which means that the ‘pores’ in the gel are large and so the stacking gel does not even inhibit the migration of large proteins. However, the amino acid residue glycine that is present in the running buffer surrounding the gels is relatively uncharged at the lower pH (6.8) of the stacking gel, thus resulting in a slow moving buffer. In contrast, the charged SDS-bound proteins in 152 Chapter the sample are able to migrate much faster through the stacking gel, causing the proteins to be compressed and ‘stacked’ into a tight band, less than mm thick, at the running front of glycine before it reaches the resolving gel. Once the running buffer reaches the resolving gel, glycine is charged at the higher pH (8.8) and migrates swiftly through the gel. The mobility of the proteins is thus solely dependent on their mass here. The resolving gel can be of different “percentage” strength (e.g. 8%, 10%, 15% etc.) depending on the concentration of acrylamide present in the resolving gel mixture. High percentage gels contain a high concentration of acrylamide, resulting in small ‘pores’ in the gel. Consequently, low molecular weight proteins are better separated as they are able to migrate through the gel better than the larger proteins. Silver staining is chosen as the method of staining as it is more sensitive and allows protein spots containing 10-100 nanograms of protein to be easily seen. Generally in silver staining, silver ions bind to the proteins in the gel, and are reduced to metallic silver, causing the protein bands in the gel to be visualized [21]. This method forms the basis of the Silver Stain Plus kit (Bio-Rad laboratories), which was used for staining in this study. In this method, the proteins are first fixed in the gel with a solution containing methanol, acetic acid, and glycerol. The gels are then soaked in a solution containing a silver-ammine complex bound to colloidal tungstosilicic acid. Silver ions transfer from the tungstosilicic acid to the proteins in the gel by means of an ion exchange or electrophilic process. Formaldehyde in the alkaline solution reduces the silver ions to metallic silver to produce the images of protein bands or spots. In this study, a µL sample loading volume in a 10% resolving gel SDS-PAGE was first attempted (Figure 7.5). 153 Chapter Figure 7.5 Plasma and Serum protein bands in SDS-PAGE gel (10% resolving gel with µl of loaded sample). The healthy samples are depicted by “H prefix”, while cancerous samples are depicted by “C prefix”. The SDS-PAGE gel using 10% resolving gel and µL of sample loading volume (Figure 7.5) does not present any significant result. In fact, the 26kDa LG3 protein band cannot be seen in these parameters. These indicate that the loaded protein volume is too low, resulting in lower concentration proteins not being stained during the silver staining process. As such, another attempt on SDS-PAGE was done using 10% resolving gel and 15 µL of loading sample volume (Figure 7.6). 154 Chapter Figure 7.6 Plasma and Serum protein bands in SDS-PAGE gel (10% resolving gel with 15 µl of loaded sample). The healthy samples are depicted by “H prefix”, while cancerous samples are depicted by “C prefix”. The red arrows correspond to the LG3 protein band. In Figure 7.6, the 26kDa LG3 protein band is visible, but barely, due to too much background staining. This is most likely due to the poor technique and/or contamination during the silver staining process. With this SDS-gel, it would be impossible to compare accurately the LG3 intensity differences in healthy and cancerous samples. In a bid to further improve the quality of the separation, SDSPAGE with 12% resolving gel and 15 µl of loading sample volume were attempted. 155 Chapter Figure 7.7 Plasma and Serum protein bands in SDS-PAGE gel (12% resolving gel with 15 µl of loaded sample). The healthy samples are depicted by “H prefix”, while cancerous samples are depicted by “C prefix”. The red arrows correspond to the LG3 protein band. The corresponding SDS-PAGE not only showed a clear visible 26kDa LG3 protein band, it also reflected the band’s intensity differences between healthy and cancerous samples. This result clearly supported earlier research [22] that the concentration of endorepellin is down-regulated significantly in cancer patient’s body fluids and therefore serves as a potential biomarker. 7.4.3 Atomic force microscopic study AFM is a versatile tool, which allows investigating the protein–protein interactions from different perspectives. In this study, the interaction between LG3 and its associated antibody is important evidence for its magnitude of presence. The interaction is highly specific and possesses a high degree of spatial and orientation specificity. Several substrates were used for the protein complex detection from AFM 156 Chapter topographies. Mica substrate is most commonly used for protein AFM imaging because of its hydrophilic character, atomically flatness and it provides high affinity for proteins [22, 23]. As a blank, a freshly cleaved mica substrate was probed by atomic force microscopy (Figure 7.8 (A)). The average roughness of this substrate determined by AFM image, recorded under contact mode in air with a commercial silicon nitride cantilever was found to be about 0.07 nm across an area of µm × µm. According to the size of the proteins (~85-KDa) to be studied, mica is suitable for the observation of such molecule. The AFM experimental approach reported in this work is based on the comparison between the AFM topographies and height histograms of antibody mica surfaces before and after incubation with specific antigens. The height histograms display a peak, its position is assumed to average height of the structures on the surface. In the first step of our experimental procedures, imaging of antibodies absorbed to mica was preceded. Samples were prepared by passive adsorption antiHSPG2 to freshly cleaved mica surface. Thus, antibodies were diluted in PBS in a concentration of about µg mg-1. Figure 7.8 (A) shows the image of mica substrate before any absorption. Figure 7.8 (B) shows anti-HSPG2 molecules on mica imaged in contact mode. Most of the anti-HSPG2 molecules are isolated and moderately homogenously distributed on mica when the concentration is µg mg-1 and incubation time is 30 min. In addition, few anti-HSPG2 aggregates formed of two or more molecules were present. The aggregate formation can be controlled by optimization of exposure time and pH conditions. Furthermore, the height histogram 157 Chapter of a section analysis reveals the single molecules and aggregates in a topographic images. In the second step, the above mica pre-absorbed antibodies have been exposed to control, tumor plasma and serum samples solution and the resulting surfaces were probed. Figure 7.8 (C) shows antigen antibody complex formation during the adsorption of antigens on the antibody adsorbed mica surface. Figure 7.8 (B) and (C) did not exhibit the same surface morphology reflecting that an increase of the surface coverage after the exposure of the control as well as tumor samples. This trend observed all the samples studied. This may be owing to the stronger binding of antibodies on substrate after complexation. Histogram peak measurements of both antibody and antibody–antigen complex surface, provides the evidence for complexation. When compare the histograms of antibody adsorbed substrate (peak width 26 nm and height nm) and antibody-antigen complex surface (peak width 43 nm and height nm) showed the possible complexation under optimized probing conditions. Even though, antibodies formed aggregates on the substrates (peak width ~50 to 53nm and height ~4 nm) there is no double complexation observed in complexed surfaces. The same probing conditions were used for control samples. Figure 7.8 (D) shows the topographic image of antigen-antibody complexation from control plasma samples. The image showed the complextion of single antibody and antigen molecules, in addition, double and few triple complexation also observed in healthy control plasma samples. This outcome was further confirmed by the surface histographic analysis. The peak measurement for double complex (Peak width ~80 158 Chapter nm) and triple complex (Peak width ~110 nm) proved the possible aggregate complexation. Similar trend was observed in all control samples studied. The possible reason for this difference in complexation for tumor and control samples might be the concentration of endorepllin. In the excess of endorepellin availability in healthy control plasma, the antibodies tend to form aggregate complexation. This phenomenon supports the results observed in SDS-PAGE analysis. (A) (B) (C) 159 Chapter (D) Figure 7.8 (A) AFM image of mica substrate before absorption. (B) AFM image of anti-HSPG2 molecules on mica imaged in contact mode. (C) AFM image of antigenantibody complex from tumor plasma sample (concentration mg mg-1). (D) AFM image of antigen-antibody complex from control plasma sample (concentration mg mg-1). 7.5 Conclusion An attempt was successfully made to apply atomic force microscopy to compare the levels of endorepellin expression in both tumor and control samples. To identify and understand the biological activities and chemistry involved, computational modelling of the protein was carried out. Conventional proteomics were performed in this study in a bid to differentiate endorepellin expression in the tumour and control samples. Through the Bradford assay, it was apparent that the total protein concentration for both healthy and cancerous samples was similar and fall within a range of 69 mg mL-1 to 75mg mL-1. Protein profiling was carried out using one-dimensional polyacrylamide gel electrophoresis (1D SDS-PAGE) and LG3 was successfully found to be less expressed in cancerous plasma and serum than in healthy samples. 160 Chapter 7.6 References [1] R.W. Risebrough, W. Walker Ii, T.T. Schmidt, B.W. De Lappe, C.W. Connors, Nature 264 (1976) 738. [2] J. Dachs, R. Lohmann, W.A. Ockenden, L. Méjanelle, S.J. Eisenreich, K.C. Jones, Environ. Sci. Tech. 36 (2002) 4229. [3] J.F. Brown Jr, Environ. Sci. Tech. 28 (1994) 2295. [4] D.L. Phillips, A.B. Smith, V.W. Burse, G.K. Steele, L.L. Needham, W.H. Hannon, Arch. Environ. Health 44 (1989) 351. [5] D.G. Patterson Jr, L.Y. Wong, W.E. Turner, S.P. Caudill, E.S. Dipietro, P.C. McClure, T.P. Cash, J.D. Osterloh, J.L. Pirkle, E.J. Sampson, L.L. Needham, Environ. Sci. Technol. 43 (2009) 1211. [6] S. Freels, L.K. Chary, M. Turyk, J. Piorkowski, K. Mallin, J. Dimos, H. Anderson, K. McCann, V. Burse, V. Persky, Chemosphere 69 (2007) 435. [7] S. Harrad, C. Ibarra, M. Robson, L. Melymuk, X. Zhang, M. Diamond, J. Douwes, Chemosphere 76 (2009) 232. [8] R.F. Herrick, M.D. McClean, J.D. Meeker, L.K. Baxter, G.A. Weymouth, Environ. Health Perspect. 112 (2004) 1051. [9] R.F. Herrick, J.D. Meeker, R. Hauser, L. Altshul, G.A. Weymouth, Environ. Health 64 (2007) 224. [10] M. Kohler, J. Tremp, M. Zennegg, C. Seiler, S. Minder-Kohler, M. Beck, P. Lienemann, L. Wegmann, P. Schmid, Environ. Sci. Tech. 39 (2005) 1967. [11] K. Norström, G. Czub, M.S. McLachlan, D. Hu, P.S. Thorne, K.C. Hornbuckle, Environ. Int. 36 (2010) 855. [12] E. De Felip, A. Di Domenico, R. Miniero, L. Silvestroni, Chemosphere 54 (2004) 1445. [13] J.D. Meeker, S.A. Missmer, L. Altshul, A.F. Vitonis, L. Ryan, D.W. Cramer, R. Hauser, Environ. Health (2009) 32. [14] M. Beclayan, W.H.Roos, G.J.L. Wuite, Mol.Cel.Proteomics. (2010) 1678. [15] J. Mes, L. Marchand, D.J. Davies, Environ. Tech. 11 (1990) 747. [16] Z.W. Polishuk, D. Wassermann, M. Wassermann, Environ. Res 13 (1977) 278. [17] C. Weistrand, K. Norén, A. Nilsson, Environ. Sci. Poll. Res. (1997) 2. 161 Chapter [18] R. Timpl, D. Tisi, J.F. Talts, Z. Andac, T. Sasaki, E. Hohenester, Matrix Biol. 19 (2000) 309. [19] K. Carroll, K. Ray, B. Helm, E. Carey, Electrophoresis 21 (2000) 2476. [20] M. Mongiat, S.M. Sweeney, J.D. San Antonio, J. Fu, R.V. Iozzo, J. Biol. Chem. 278 (2003) 4238. [21] C.R. Merril, M.L. Dunau, D. Goldman, Anal. Biochem. 110 (1981) 201. [22] J.W. Chang, U.B. Kang, D.H. Kim, J.K. Yi, J.W. Lee, D.Y. Noh, C. Lee, M.H. Yu, Proteo.Clinical Appl. (2008) 23. [23] C. R. Merril, M.L. Dunau, D. Goldman, Anal. Biochem. 110 (1981) 201. 162 [...]... separation of proteins is dependent almost entirely on the differences in relative molecular mass of polypeptides The speed of migration is dependent on the size of the ‘pores’ in the gel mesh which is in turn determined by the percentage of acrylamide present in the gel The polyacrylamide gel consists of 2 parts: the stacking gel and the resolving gel The stacking gel is used to form the wells in which the. .. better separated as they are able to migrate through the gel better than the larger proteins Silver staining is chosen as the method of staining as it is more sensitive and allows protein spots containing 10-100 nanograms of protein to be easily seen Generally in silver staining, silver ions bind to the proteins in the gel, and are reduced to metallic silver, causing the protein bands in the gel to be visualized... temperature for a specified period (60 min, 30 min) of time The amount of antibody binding depends on several factors, including degree of washing with water, concentration of antibody, and other incubation conditions The conditions of binding with surface adsorbed protein are summarized in Table 7. 3 In the control experiments, Anti-HSPG2 was used with the same incubation times and at the same concentrations... coordinating residues The values on the dotted yellow lines depict the distance between the 2 species in angstroms 7. 4.2 Proteomic study 7. 4.2.1 Total protein quantification in samples using the bradford assay The total protein concentration in each of the plasma and serum samples to be used in this study must be known so that in the subsequent stage of protein separation by SDS-PAGE, the same amount of. .. SDS-PAGE 7. 3.2.3.1 Assembly of apparatus before casting the polyacrylamide gels To prepare for casting the polyacrylamide gels, the 0 .75 mm spacer and short plates were cleaned with 70 % ethanol The cleaned 0 .75 mm spacer and short plates were then inserted into the casting frame, placed on the gasket and held together by the casting stand 143 Chapter 7 7.3.2.3.2 Preparation of SDS-PAGE SDS-PAGE gels... the charged SDS-bound proteins in 152 Chapter 7 the sample are able to migrate much faster through the stacking gel, causing the proteins to be compressed and ‘stacked’ into a tight band, less than 1 mm thick, at the running front of glycine before it reaches the resolving gel Once the running buffer reaches the resolving gel, glycine is charged at the higher pH (8.8) and migrates swiftly through the. .. containing 40% methanol and 10% acetic acid (v/v) overnight on an 145 Chapter 7 UltraRocker Rocking Platform at room temperature This was followed by rinsing and washing the gels in deionised distilled water for 30 min Gels were then stained in developer solution until desired staining intensity was reached, and placed in 5% acetic acid for 30 minutes to stop the reaction The gels were then scanned using... protein sample is loaded It has a very low acrylamide concentration, which means that the ‘pores’ in the gel are large and so the stacking gel does not even inhibit the migration of large proteins However, the amino acid residue glycine that is present in the running buffer surrounding the gels is relatively uncharged at the lower pH (6.8) of the stacking gel, thus resulting in a slow moving buffer In. .. that the resulting sample had a protein concentration of 1 μg/μl The samples were then heated at 99oC for 7 minutes in a thermo-cycler, and then centrifuged at 13,000 RPM for 10 minutes 5 µl of protein standard (Precision plus protein All blue® standards), control (2 x loading buffer) and samples were loaded into the wells once the SDS-PAGE gels had polymerized A constant voltage 144 Chapter 7 of 100V... persulphate 7. 3.2.3.4 Gel staining The proteins that have been separated on gels can be made visible by staining them with dyes or metals A number of different protein stains exist like the Coomassie Blue stain, Ruby fluorescent stain and Silver stain Each type of stain has its own characteristics and limitations with regard to the sensitivity of detection and the types of proteins that stain best[19] In this . cancer cells, but also in the blood and other body fluids into which these proteins are secreted. This can therefore aid in the identification of a normal cell transforming into a cancerous state protein by identifying its protein-antibody complex. In this present study, the structure and environment of the LG3 domain in the endorepellin was identified using homology modelling and the. factors, including degree of washing with water, concentration of antibody, and other incubation conditions. The conditions of binding with surface adsorbed protein are summarized in Table 7. 3. In