Grain protein composition in a hurry

The protein composition of a seed contains a wealth of information about its identity (variety and species), its potential for processing quality and even about its genetic origins. This information can have great commercial benefit when food grains are delivered after harvest, but this benefit is only fully realised if the information is provided within a short time - preferably within several minutes - in time for decisions to be made about where a truckload of grain should be dumped.

For wheat, varietal identity is a major item of information needed on delivery after harvest, as this serves to indicate the many grain-quality attributes that have been 'built in' by the breeder in selecting the variety. For any specific class of grain, there is a select range of varieties prescribed. In addition, extra premiums are now paid for the delivery of a few special 'Golden Rewards' varieties.

There is, thus, the urgent need for on-the-spot identification of variety at the grain-receival site ('elevator' or ' silo'), when grain must be assessed on delivery for variety within a few minutes with minimal facilities. At present, this issue is accommodated by taking a legal declaration of varietal identity from the truck driver, plus sub-sampling the load delivered for subsequent analysis. However, if the declaration of identity proves subsequently to be wrong, it is already too late to avoid the admixture of the inappropriate load with the rest of the grain.

This verification of identity in the sub-sample is performed at a central laboratory, generally on the basis of protein composition by gel electrophoresis or HPLC. Such analyses have proved to be effective though slow, requiring significant time, extensive backup of lab resources, plus operator expertise.

The Agilent lab-on-a-chip equipment has introduced a breakthrough in methodology for determining grain-protein composition quickly in commercial situations, such as at the grain silo. The equipment is relatively small and thus portable (Figure 1). It requires no more backup than electric power, but a small centrifuge is also needed for the preliminary step of extracting proteins from the crushed grain samples.

For variety identification, we have found the most effective procedure is to extract the full complement of grain polypeptides with a combination of 1% SDS and 1% DTT (Uthayakumaran et al, 2005). The detergent SDS (sodium dodecylsulfate) coats the proteins, assisting in their extraction and in their size-based fractionation. The DTT (dithiothreitol) assists further in protein extraction by breaking the disulfide bonds between the polypeptide chains of the very large glutenin polymers of gluten.

Ten such extracts can be loaded onto one Lab-Chip (together with reagents) for rapid capillary electrophoresis in the Bioanalyzer. The production of each profile takes only about 50 seconds to complete (Figure 2, top), with the profile appearing immediately on the attached computer screen. The integral software transforms the elution profile into a set of electrophoretic bands (Figure 2, bottom). The gel patterns simulate the traditional SDS-PAGE patterns, being preferred by many operators because of familiarity and the ease of comparison across a set of samples.

The smallest proteins are eluted first, appearing at the left of the elution profiles, but at the bottom of the simulated gel patterns. A ladder of standard proteins is analysed with each set of 10 samples, providing a basis for estimating apparent molecular weights and importantly, for ensuring reproducibility. Multiple analyses of the same grain extract have demonstrated excellent reproducibility, yielding less than 1% relative standard deviation (RSD); multiple extractions of the same sample show less than 2% RSD.

Each variety of wheat produces a distinctive and characteristic profile (or set of bands). Normal procedure would thus involve side-by-side comparison of the delivered grain sample with an authentic sample of the variety declared by the truck driver. Distinction has proved to be possible for most of over 40 wheat varieties commonly grown in Australia. Preliminary analyses indicate the likelihood that the same procedure can be applied to a wide range of grain species.

Beyond the need to verify varietal identity, the Lab-Chip profiles also provide information about the genetic potential of the grain for dough properties. The polypeptides resolved in the upper half of the gel patterns are the high-molecular-weight subunits of glutenin, whose composition is closely related to dough properties. Analysis of the subunits present offers the additional advantage of obtaining a prediction of this important aspect of wheat quality, but there is the further need to assess the possible extent to which growth conditions may have modified this genetic potential.

The lab-on-a-chip version of capillary electrophoresis promises to overcome the present difficulties of identifying variety and dough-type, thereby offering a platform for quickly checking grain quality beyond the confines of the traditional laboratory. The potential of the method for deployment beyond the laboratory was tested successfully in the recent wheat harvest by using the equipment at silos in southern NSW and Victoria. Some hundreds of delivery samples were analysed for varietal identity. Most were found to be correct, as declared, but some were found to be incorrect. This approach to "real-time" analysis of protein composition was thus shown to fulfill its potential.

Further information about the Lab-Chip and the Bioanalyser can be obtained from Agilent Technologies, www.agilent.com