Gluten Analysis

Abstract

Wheat, barley and rye are sources of gluten and diverse food products are made from the grains of these cereals. Despite some species-specific differences, the molecular properties of the gluten proteins show similar characteristics in forming a unique protein network that has been extensively described in terms of its subunits and composition, its function in bakery products and its implications for human health.

There are many reasons for analysing gluten to serve purposes as diverse as assessing flour quality, selecting and breeding suitable cereal varieties, identifying varieties, identifying the source of gluten in a product, and quantifying gluten in food and drink, especially to protect gluten intolerant consumers.

The level of gluten in food and drink deemed to be safe for people with coeliac disease or non-coeliac gluten sensitivity is set in legislations. Various systems are in place worldwide to regulate food labelling and various testing methods are used or are available to quantify gluten, but cross-border standardisation to harmonize the quantification of gluten in food products has yet to be agreed.

To analyse gluten, the proteins must be separated from other possibly interfering food components. As it is difficult to solubilize gluten, gluten extraction is a critical part of the process. The level of gluten, the type of food matrix and the available technology impose further limitations and challenges.

In this chapter, we describe a strategy to select the most suitable gluten analysis approach according to the purpose, sample type, gluten level and performance characteristics required. The advantages and disadvantages of qualitative and quantitative gluten analysis techniques, such as gel electrophoresis, immunoassays, asymmetric field flow fractionation multi-angle laser light scattering, chromatography and chromatography coupled methods are covered.

Appendices

Appendix I

SDS-PAGE protocol used by the Wheat Chemistry and End–Use Quality Laboratory of CIMMYT

Reagents and main steps of the procedure were published in Peña et al. (2004, revised in 2018).

PREPARATION OF REAGENTS

Reagent 1. 1 M Tris pH 8.5

Dissolve 30.3 g of Tris in 220 ml of distilled water. Adjust pH to 8.5 with concentrated HCl. Bring the total volume up to 250 ml with distilled water. Store in the refrigerator.

Reagent 2. 1 M Tris pH 6.8

Dissolve 12.1 g of Tris in 64 ml of distilled water. Adjust pH to 6.8 with concentrated HCl. Bring the total volume up to 100 ml with distilled water. Store in the refrigerator.

Reagent 3. 10% sodium dodecyl sulfate (SDS)

Dissolve 5 g of SDS in 40 ml distilled water and bring the total volume up to 50 ml with distilled water. Store at room temperature (20–25 °C).

Reagent 4. Stock acrylamide solution (40%T and 1.3%C) for running and stacking gels

Weigh 0.52 g of bis-acrylamide and 39.5 g of acrylamide. Dissolve in approximately 70 ml of distilled water and bring the total volume up to 100 ml. Homogenize and filter the solution before use. Store in a dark (translucent) container in the refrigerator.

CAUTION: use gloves and mask when working with acrylamide.

Reagent 5. Stock sample buffer for total protein extracts

Dissolve 12 g of glycerol in 36 ml of distilled water. Add 0.76 g of Tris, 4.0 g of SDS, 750 mg of dithiothreitol (1.5% DTT) and 6.0 mg of bromophenol blue. Adjust to pH 6.8 with concentrated HCl and bring the total volume up to 50 ml with distilled water.

Reagent 6. Tris-glycine stock for running buffer

Put 30.0 g of Tris in a 1-L beaker. Add 887 ml of distilled water and stir. While stirring the solution first add 144 g of glycine then 10 g of SDS. The pH of the solution should be 8.3. If the pH is higher, do not adjust it with HCl as it would alter the desired ionic strength.

Reagent 7. Staining and destaining solutions

Solution 1 is a 12% trichloroacetic acid solution. Dilute 120 g of trichloroacetic acid in distilled water and make up to 1 L.

Solution 2 is the staining solution. Mix 400 ml of methanol, 100 ml of glacial acetic acid, 0.1 g of Coomassie Brilliant Blue R-250, and 500 ml of distilled water (1 L in total).

Reagent 8. 1.5% ammonium persulfate

Dissolve 150 mg of ammonium persulfate in 10 ml of distilled water. Prepare fresh immediately before use.

1.1 Procedures

Extraction of total proteins

Weigh 20 mg of sample into a microcentrifuge tube. Add 300 μl of sample buffer (reagent 5) and incubate for 5 min in an Eppendorf Thermomixer comfort at 90 °C and 1400 rpm. Centrifuge for 5 min at 13000 rpm in an Eppendorf Microcentrifuge 5415C (Brinkmann Instruments Inc., NY).

Gels for total protein extracts from durum wheat and bread wheat

The final acrylamide concentration to be used in the gel will depend on the size and type of bands to be examined in detail. For the running gel, 9–10% acrylamide is the most common gel concentration when examining high molecular weight glutenins from whole grain protein extracts. However, for better separation of subunits 2 and 2*, gels of around 13% acrylamide are more appropriate.

Formulas to determine gel concentrations

$$ \mathrm{Concentration}\ \mathrm{of}\ \mathrm{acrylamide}\ \left(\mathrm{T}\right)=\frac{\mathrm{g}\ \mathrm{of}\ \mathrm{acrylamide}+\mathrm{g}\ \mathrm{of}\ \mathrm{bis}-\mathrm{acrylamide}}{\mathrm{total}\ \mathrm{volume}\ \mathrm{of}\ \mathrm{solution}}\times 100 $$

$$ \mathrm{Concentration}\ \mathrm{of}\ \mathrm{bis}-\mathrm{acrylamide}\ \left(\mathrm{C}\right)=\frac{\mathrm{g}\ \mathrm{of}\ \mathrm{bis}-\mathrm{acrylamide}\kern2.78em }{\begin{array}{l}\mathrm{g}\ \mathrm{of}\ \mathrm{Acrylamide}-\mathrm{g}\ \mathrm{of}\ \mathrm{bis}-\mathrm{acrylamide}\\ {}\end{array}}\times 100 $$

Staining and destaining gels

Submerge the gels for 5 min in a 12% trichloroacetic acid solution (reagent 1), then place them in staining solution (solution 2) for 4 h, and finally for destaining place them in distilled water for 24 h.

Reagents for glutenin and gliadin extracts

Solution 1.1. 0.08 M Tris-HCl buffer pH 8.0

Weigh 4.8 g of Tris and 20 g of SDS and dissolve them in 500 ml of distilled water, adjusting the pH to 8.0 with HCl.

Solution 1.2. 50% propanol

Solution 1.3. Mix 500 ml of each of solution 1.1 and 1.2

Solution 1.4. For 10 ml of solution 1.3, mix in 200 mg of DTT. This reagent should be prepared the same day.

Solution 1.5. For 10 ml of solution 1.3, add 140 μl of 4-vinyl-pyridine and mix. This reagent should be prepared the same day.

Solution 1.6. Glutenin extraction buffer

2% SDS (w/v), 40% glycerol (w/v), 0.02% (w/v) bromophenol blue.

Dissolve 20 g of glycerol in 36 ml of distilled water. Add 0.76 g of Tris, 1.0 g of SDS, and 10.0 mg of Coomassie Brilliant Blue R. Adjust the pH to 6.8 with concentrated HCl. Make the volume up to 50 ml with distilled water.

Solution 1.7. Gliadin extraction buffer

2% SDS (w/v), 40% glycerol (w/v), 0.02% (w/v) bromophenol blue. Dissolve 20 g of glycerol in 36 ml of distilled water. Add 0.76 g of Tris, 1.0 g of SDS, and 10.0 mg of Coomassie Brilliant Blue R. Adjust the pH to 8.0 with concentrated HCl. Make the volume up to 50 ml with distilled water.

Running gel (For two 15%T gels, 17 cm high and 1 mm thick)

• 24.7 ml of Tris pH 8.5 (Reagent 1)

• 24.4 ml of Acrylamide Stock for Running Gel (Reagent 4)

• 650 μl of 10% SDS.

• 14.0 ml of Distilled Water

This mixture is deaerated with a Sonics Branson 5510 for 2–4 min. Immediately filter the mix through coarse filter paper. Add 1.3 ml of fresh 1.5% ammonium persulfate (reagent 8). Assemble frames for pouring gels then add 50 μl of TEMED to the mixture. Mix gently for 5 seconds and pour the gel solution into the frames. Immediately apply 1 ml of butyl alcohol to the top of the gel to prevent the formation of a meniscus. The alcohol should be applied slowly with a syringe.

Stacking gel (For two 4.8%T gels, 3.0 cm high and 1.0 mm thick)

Before adding the stacking gel solution, remove the butyl alcohol from the top of the running gel.

• 6.12 ml of Distilled Water

• 1.10 ml of Tris pH 6.8 (Reagent 2)

• 1.05 ml of acrylamide stock for stacking gel (reagent 4).

• 90 μl of 10% SDS.

• 380 μl of 1.5% ammonium persulfate

Mix reagents together gently, add 50 μl of TEMED, and mix again. Apply to the top of the running gel, being careful that there are no bubbles in the lanes of the Teflon lane former (comb). This can be achieved by inserting the Teflon comb at an angle of approximately 30°. Let the gel stand for 30 min to 1 hr. Very carefully remove the Teflon comb and with a syringe remove the excess solution which did not polymerise. Fill the lanes with running buffer (see below).

Running buffer

Mix 400 ml of stock for running buffer (reagent 6) and 3.6 L of distilled water. Use approximately 2.5 L of buffer per tank (e.g. Protean BIO-RAD equipment, Bio-Rad Laboratories, Richmond, California) for 2 gels (4.5 L for a buffer tank for 6 gels).

Running the gel

Load the samples in each lane (4–8 μl of extracted protein). Assemble the equipment, connect the hoses of the cooling system (15 °C) to running water and turn on the power. The running time depends on the current (mA) and the porosity (%T) of the gel. Electrophoretic separation can be run at 12.5 mA per gel for about 19 h (overnight). When using more than 25 mA per gel (runs of approx. 8 h), it is necessary to use a cooling apparatus to maintain temperature at around 15 °C. For a shorter running time of 4–5 h, run the electrophoresis at 35–40 mA per gel at 15 °C until the colored line arrives at the bottom edge of the gel. In order to maintain ionic strength, it is recommended to use a small pump connecting the lower to the upper buffer containers to recirculate the running buffer.

Preserving gels

Fresh gels may be kept for limited time if placed in polyethylene plastic bags in the refrigerator. Drying the gels with a gel drier is more convenient to preserve the gels for longer.

Selective extraction of gliadins and glutenins for SDS-PAGE

According to Singh et al. (1991) with modifications.

Appendix II

A-PAGE protocol for gliadin analysis

The main steps are described in Metakovsky and Novoselskaya (2001) for gels of standard size (18 ⨯ 20 cm). For long acrylamide gels (18 ⨯ 32 cm) it is advisable to use the protocol previously described by Branlard et al. (1990).

Reagents

• 1. 70% ethanol

• 2. 1.5% aluminium lactate pH 3.1

  • Dissolve 15 g of aluminium lactate in 800 ml of distilled water∗. Adjust pH to 3.1 with lactic acid. Bring the total volume to 1 L with distilled water.

• 3. 1% ascorbic acid

  • Dissolve 0.1 g of ascorbic acid in 10 ml of distilled water. Prepare fresh for each use.

• 4. 1% Fe(SO₄)₃

  • Dissolve 0.1 g of Fe(SO₄)₃ in 10 ml of distilled water.

• 5. 40% acrylamide

  • It is highly advisable to purchase 40% acrylamide solution ready to use. Alternatively, dissolve 40 g of high quality acrylamide in 100 ml of distilled water. Store at 4 °C.

• 6. 2% bis-acrylamide.

  • It is highly advisable to purchase 2% bis-acrylamide solution ready to use.

  • Alternatively, dissolve 2 g of high quality bis-acrylamide in 100 ml of distilled water. Store at 4 °C.

• 7. 0.33% hydrogen peroxide

  • Purchase 3% hydrogen peroxide (10 volumes) from a pharmacy or store and keep it at 4 °C. Make 1.1 ml of 3% hydrogen peroxide (10 volumes) up to 10 ml with distilled water. Prepare fresh for each use.

• 8. Stock sample buffer

  • Dissolve 3 ml of glycerol in 1 ml of 1.5% aluminum lactate pH 3.1. Add a small portion (a few grains of powder) of pyronin or methyl green. Bring the total volume up to 10 ml with distilled water.

• 9. Stock running buffer (0.15% aluminium lactate pH 3.1)

  • Dilute 100 ml of 1.5% aluminum lactate with 900 ml of distilled water.

∗Dr. Metakovsky consistently uses distilled water. The classic ultra-pure water influences the resolution of the gliadins and may alter the mobility of some gliadin alleles. Twice-deionized water (today referred to as ultra-pure water) was used by Branlard et al. (1990).

Procedures

Gel preparation

8.3% Acrylamide, 0.42% Bis-Acrylamide, 0.1% Ascorbic Acid, 0.00067% Fe(SO₄)₃, 0.15% Aluminum Lactate, pH 3.1

The glass plates (20 ⨯ 20 cm and 2 mm thickness for a normal gel or 30 ⨯ 20 cm and 2 mm thickness for a long gel for higher resolution) are cleaned with 70% ethanol then by spreading droplets of glycerol until a thin film forms and drying with KimWipes. This treatment makes it easier to remove gels from plates.

Mix 21 ml of 2% bis-acrylamide and 20.8 ml of 40% acrylamide. Add 67 μl of 1% Fe(SO₄)₃, 10 ml of 1% ascorbic acid, 10 ml of 1.5% aluminum lactate, and bring the total volume up to 100 ml, filter through filter paper and degas, then cool on ice before use. Add 0.4 ml of 0.33% hydrogen peroxide, mix well and pour rapidly into the glass plate assembly. The comb is inserted and the gel is polymerized for 10 min.

Gliadin extraction

Weigh about 20 mg of sample into a microcentrifuge tube. Add 150 μl of 70% ethanol and incubate for 30 min at room temperature. Centrifuge the sample for 5 min at 12500 rpm. Transfer the supernatant to a microcentrifuge tube and mix with an equal volume of the sample buffer. Freshly prepared (within two days) samples should be used to obtain well resolved electrophoretic profiles.

EXTRACTION OF GLIADINS FOR A-PAGE

Electrophoresis

After rinsing the wells with running buffer, the glass plates containing the gel are placed in the vertical slab gel apparatus. Then the wells are filled with the buffer and 20 to 30 μl of the samples are slowly loaded. Electrophoresis from the anode (the upper buffer) to the cathode (the lower buffer) is performed for 10–20 min at 220 V and then for 2.5–3 h at 550 V without buffer circulation. The lower tank of the vertical apparatus is cooled with tap water or in a cooling system at 10 °C.

Staining/destaining

The gels are kept on one of the glass plates to reduce the risk of damaging the gels. The staining procedure is the same as for the SDS-PAGE method.