Techniques for Studying Gluten Protein-Free-Samples for Students

Question: Find a protein of interest and describe five Protein Technologies that have been used to study it. Answer: Protein: Gluten Gluten comprises of two different protein components; gliadin and glutenin. It is the composite of both glutelin and prolamin proteins that are conjoined to starch in grass-related cereal endosperms. Gliadin has between 30000Da and 80000 Da of relative molecular mass while glutenin has several millions Da of relative molecular mass (Hochegger et al, 2015).Gluten is thus a long molecule with strong but flexible characteristics. Structurally, gluten is a yellowish-white, elastic, sticky and tough protein. Because of its characteristics the protein is useful in the bread-making industry because it is able to trap carbon dioxide that is released in the flour-yeast reaction giving flour the chewiness characteristics and making it rise to keep a particular shape. There are different techniques of studying gluten components and analysing their characteristics. These include R5 ELISA test kits, biosensors, Quantitative Real-Time PCR, Liquid Chromatography Tandem Mass Spectrometry, Gel- Perme ation Chromatography and Size Exclusion High Performance Liquid Chromatography. These techniques however vary in terms of sensitivity and in the particular characteristics of gluten protein that they measure. Sandwich R5 ELISA Test Gluten protein components are mainly analysed using the sandwich R5 ELISA format. This analysis technique has a high sensitivity and it is quite specific for each gluten protein (Hochegger et al, 2015). Usually, sandwich R5 ELISA method is utilized in quantifying antigens especially when they are lowly concentrated and also when they are in a sample that has a larger amount of proteins which are non-gluten (Tranquet et al, 2012). It is based mainly on the R5 antibody, and therefore uses both R5 antibody and R5 conjugated antibody, which bind to different antigen sites. R5 antibody can recognize celiac epitopes that are potentially toxic repeatedly occurring in prolamins. These celiac epitopes include QQPFP, PQPFP, QQPYP, QQQFP, QLPYP and LQPFP. The epitopes are found in toxic-celiac peptides such as Gliadin 33 mer peptide, Gliadin 25 mer peptide, and the Gliadin 26 mer peptide (Guerdrum, 2013). The R5 ELISA assay has a gliadins quantification limit of 1.56 ppm. The assay must however be combined with the cocktail extraction solution. Several studies indicate that the R5 ELISA technique has been internationally accepted to be the main gluten detection method in foods by the Codex Alimentarius Commission. However, quantifying gluten in hydrolysed foods using this approach is slightly inaccurate since there is always need for two intact epitopes to help quantify gluten content (Hochegger et al, 2015). As a result, a competitive R5 ELISA that is based on an R5 monoclonal antibody is used to give a precise quantification. The later can quantify intact and/or fragmented gluten since the technique uses just one antibody and thus just one epitope is needed for complete gluten detection. Additionally, the competitive system is considered cheaper but faster as compared to the sandwich ELISA14 system (Guerdrum, 2013). According to the Codex Alimentarius Commission, there is need to modify R5 competitive ELISA assay in order to enable hydrolysed gluten detection. While the competitive technique is not compatible with the coc ktail extraction solution; a combination of the competitive assay and UPEX solution gives complete and very accurate gluten analysis. In regard testing for gluten in wheat, barley and rye R-5 antibody ELISA technique remains the favorite approach (Guerdrum, 2013). This is because the antibody neither reacts with glutenins, nor glutelins in rye and barley. Currently, the Codex committee on protein analytical methods recommends R-5 ELISA technique for analyzing gluten content in foods. Figure 1: obtained from https://www.wgpat.com/proceeding_25th.html The sandwich Omega analytical method thus varies from the competitive sandwich R5 ELISA test. The table below shows the variation in these methods in regard to detecting and characterising gluten protein. Figure 1 above shows a bar graph of gluten levels (ppm) in samples of buckwheat detected by Sandwich R5 ELISA test just after extracting using the cocktail solution. comparison of Sandwich Omega ELISA and the Competitive Sandwich R5 ELISA Sandwich Omega ELISA(Sandwich R5 ELISA) The Competitive Sandwich R5 ELISA It uses 40% of ethanol solution in extracting proteins Needs 2 epitopes for two antibodies to bind a protein It underestimates content of protein in particularly for hydrolysed and/or partially hydrolysed barley. Is able to detect heated i.e. denatured and also the unheated proteins even at gluten levels150 ppm Has higher sensitivity versions for protein analysis Detects gliadin, a gluten component Utilise extraction mixture of provided compounds Only needs a single but specific epitope i.e. the R5 monoclonal antibody Needs just one antibody Can detect protein that is highly heat resistant and toxic at the same time, very accurately The technique overestimates the content of protein in barley It cannot be used to determine hydrolysed gluten proteins It is able to detect heated and/or unheated proteins It can recognize barley, rye and wheat gliadin with 3 ppm and detection levels and can even measure5 ppm (Hochegger et al, 2015). The technique detects gliadin, one of the components of gluten. Figure 2: A comparison of Sandwich Omega ELISA and the Competitive Sandwich R5 ELISA Biosensors There are several biosensors that are used in the detection and characterization of gluten components. Biosensors are used especially to detect gliadin presence in in gluten-free food products. The first electrochemical biosensor relies on an antibody that is raised against gliadins immunodominant epitope that has 5.5 g/L of limit of detection (Gomes de Sousa Filho et al, 2014). The second biosensor is based on anti-gliadin Fab fragments adsorption properties on gold surfaces. The limit of detection for gliadin has been assessed by impedance and shown to be 0.42 mg/L and by amperometry that showed 3.29 g/L LOD (Nirantar et al, 2014). Lately, a biosensor referred to as quartz crystal microbalance biosensor which incorporates nanoparticles of gold can detect gliadin that has 8 g/Kg60 limit of detection (Sensors and Materials, 2016). A different biosensor which uses antibody-conjugated immunomagnetic beads of anti-gliadin and/or fluorescence-dye-loaded immunoliposomal nanovesicles so as to form a sandwich, registered 0.6 mg/L limit of detection for gliadin. Quantitative Real-Time PCR Polymerase chain reaction techniques can also be used to detect, characterize and even quantify DNA of cereals containing gluten. These techniques are able to not only characterize but also can provide various cultivars and enable the selection of genotypes coding for different gluten proteins that have the best quality of making bread (Debnath et al, 2009). PCR in combination with agarose gels can be used detect the presence of wheat in oats. In other studies quantitative PCR system in combination with the agarose gels is used to detect wheat, rye and barley contamination simultaneously, in gluten-free food products. The use of agarose gels is however disadvantageous and therefore the wheat DNA detection and quantification relies purely on quantitative polymerase chain reaction (Q-PCR) (Wang et al, 2010). There is currently a Q-PCR system that is reliable and provides rapid wheat DNA quantification in gluten-free food products and even in the raw materials. Its development is based SYBR Green I fluorescent dye and the modified extraction protocol of Guanidine-HCl or Proteinase K DNA. This particular system is very specific in nature and has a high sensitivity presenting up to 20 pg DNA/mg quantification limit (Debnath et al, 2009). This system when compared with the levels of glutens prolamin that have been determined using R5 ELISA commercial technique, it is clear that except for a few foods that are hydrolysed and/or highly processed; every sample that has prolamin levels that are above the quantification limit of R5 ELISA test (1.5 mg/kg) gives positive signals on Q-PCR system (Meral, 2016). It is therefore recommended that this particular Q-PCR system can be relied on to confirm the presence of gluten or generally wheat in foods, as a non-immunological tool, through the DNA pathway. This is especially useful in approving foods for celiacs and for those that have gluten allergy. Liquid Chromatography Tandem Mass Spectrometry The LC-MS/MS technique is also one of the methods for studying gluten proteins. The technique can be able to detect various species based on several markers with several confirmation points. This makes it less vulnerable to provide false positives and/or false negatives while giving far more detection confirmation (Lock, 2013). Since the LC-MS/MS technique is very specific in nature, it can distinguish species through the use of multiple peptide markers. LC-MS/MS mainly differs from R5 ELISA technique since it can rely on specific markers in wheat, oats, and rye. The individual markers for the above species can distinguish one from the other through LC-MS/MS. The LC-MS/MS tool is thus advantageous than the R5 ELISA technique in that it can use a number of peptide markers with a similar number of MRMs for every gluten peptide to detect gluten presence in a particular sample with different species (Liang et al, 2011). It is therefore the most feasible approach in detecting gluten in fo ods. It has also been proven that ELISA test kits fail to detect allergens in processed foods because of processing which brings changes on the proteins structure and thus preventing the binding of an antibody; leading to false negatives The LC-MS/MS technique detects 510 ppm gluten levels in gluten-free foods provides a linear response that is linear and also larger when compared to the response which is usually obtained ELISA tests(Liang et al, 2011). The current LC-MS/MS technique involves 80-fold sample dilution; that is then injected onto LC-MSMS system. This enables the system to potentially detect low gluten levels (0.55 ppm) particularly when the concentrate is collected and the peptide markers purified using under the SPE protocol (Creese Cooper, 2007). The presence of several markers for every variety of gluten and the ready availability of scans of MRM triggered product ion gives many gluten contamination confirmation points. This also provides confidence in gluten detection results obtained while reducing false positive and/or false negative risks that emerge in ELISA tests. Gel-Permeation Chromatography Size Exclusion High Performance Liquid Chromatography Gel-permeation chromatography relies on the principle of using materials that contain dextran to separate different macromolecules according to their molecular size differences. The procedure basically determines protein molecular weights and can also be used in decreasing concentration of salt in solutions of proteins (Bacskay et al, 2014). The gel- permeation column has a stationary phase which consist small-pored inert molecules. A solution containing various molecules that have varying dimensions, are continuously passed through the column at a flow rate that is constant. Molecules which are larger than the pore sizes are not able to permeate into the gel particles but retained within a restricted area between particles. The larger molecules can pass through the spaces that are between the porous particles to rapidly move inside the column (Jannah, 2015). On the other hand, smaller molecules diffuse into the pores as they get smaller, these molecules leave this column but with a longer retention time. The column material which is frequently used in gel permeation chromatography columns is the Sephadeks G type. Other column materials in this technique include agorose, dextran and polyacrylamide. Studies have indicated that conventional SE chromatography as described above has a lot of disadvantages as it is slow and usually has column beds that are unstable. Thus, results are likely to be unreliable and difficult to quantitate. As a result, there are Size-exclusion High Performance Liquid Chromatography (SE-HPLC) columns which are not only stable but uniform and reliable and thus are utilized in analysing proteins in cereals including gluten (Bacskay et al, 2014). The SE-HPLC separations are usually fast and this allows larger sample numbers for analysis. SE-HPLC has over time been utilized extensively in analysing cereal proteins. The SE-HPLC supports have different and/or varying porosity just like the conventional gel approach. An example of the Toya-Soda-made columns includes the TSK-4000SW, the TSK-3000SW and further, the TSK-2000SW columns. These columns have approximately 450, 240 and 130 A of pore size respectively (Creese Cooper, 2007). They are thus very useful in analysing high, intermediate and even low molecular weighted proteins. The process uses columns of measure 500 X 7.5 mm i.d. with 7-8 ml exclusion volumes and approximately 22 ml of total exclusion volume. Along the process, a guard column for TSK-3000SW with measures of 100 X 7.5 mm i.d. is used (Bacskay et al, 2014). A common solvent used in SE-H PLC particularly for cereal proteins like gluten; is 0.1 M sodium phosphate, with a pH of 7.0 and contains 0.1% SDS. The SDS in the solvent binds to proteins and solubilizes them in a way that the resultant protein-SDS complex sizes relate to protein MW. In the SDS presence, TSK-4000SW separates 10,000 to 1,000,000 MW proteins while TSK-3000SW usually separates 10,000 to 200,000 MW proteins (Tranquet et al, 2012). For proteins and/or peptides that have low molecular weight, the TSK-2000SW column is used. The flow rates in the columns are maintained at 1.0 ml/minute while the analyses carried out at room temperature. In the final analyses , the log MW plotted against the time of elution gives a straight line. Its equation is then used to approximate the MW of any other unknown proteins (Taddei et al, 2013). Usually, the standard measures are run on a daily basis and they are used to inform computer program updates on elution times, percentages, MW for every peak among others. Analysis of Gliadins using the SE HPLC Whole gliadin sample when tested shows the major peak for , and gliadins that correspond to 28,000 Molecular Weight. It also shows smaller peaks and/or shoulders which correspond 11000MW, 41000MW, 63000MW of ? gliadins and further 105000 MW high-molecular-weight gliadin (Tranquet et al, 2012). SE-HPLC is also used in comparing gluten proteins in native and/or reduced forms. For example, the high-MW gliadin is made up of intermediate MW heterogeneous oligomers. Its Molecular weight lies between gliadin and glutenin: and has an average MW of about 125,000 (Bacskay et al, 2014). The SE-HPLC technique rapidly confirms that the high-MW gliadin protein consists mainly, subunits of low-MW glutenin which are joined by way of disulphide bonds. SE-H PLC technique is useful in making comparison between proteins whether in their native or reduced states. Conclusion Gluten protein is a major requirement in bread making. Its levels must however be monitored especially understanding that it can affect a section of the population that are susceptible to celiac disease and those that have gluten allergy. This discussion highlights each technique that is relied upon to detect the amounts of gluten components in foods for rectification purposes. International companies that also sell wheat, barley, oat, rice and rye products rely on the technologies discussed to ensure that their products are gluten free. All the techniques discussed including sandwich R5 ELISA test kits, biosensors, Quantitative Real-Time PCR, Liquid Chromatography Tandem Mass Spectrometry, Gel- Permeation Chromatography and Size Exclusion High Performance Liquid Chromatography; is crucial in the food production sector. 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