New Frontiers in the Analysis of GMO Crop Proteins

By Glenn Petrie, Ph.D.
Senior Scientific Advisor
ABC Laboratories
www.abclabs.com

Genetically Modified Organisms (GMO) have been on the market for over two decades. These plants have been engineered for a variety of properties including:

  • Herbicide resistance

  • Cold/heat tolerance

  • Disease resistance

  • Increased yield

  • Improved quality

  • Pest resistance

Either internally or through licensing agreements large Agro Science companies have combined many of these properties into a single species. This may result in the introduction of 10-20 modified proteins. Each protein is present at different levels in each plant tissue and these levels typically change within the lifespan of the crop. The public concern with GMO crops has led to stringent control. The licensing requirements require careful control of the plants (particularly seeds), multiple field trials and careful monitoring of the modified plant proteins.

This presents quite an analytical challenge: 10-20 modified proteins in up to 12 different plant tissues. Sample preparation alone presents a daunting task. Plants are separated into their component tissues and each tissue macerated (multi-step), often lyophilized and ground to a fine homogeneous powder. The proteins are extracted, typically requiring different extraction methods depending upon the particular protein or tissue.

Once prepared and extracted actual quantitation of the proteins is required. The technique must be sensitive (low ng/mL), specific (thousands of proteins) and precise (crops are sampled several times during their lifecycle). Based on these requirements the methods available are:

  • ELISA

  • Western

  • LC/MS/MS

ELISAs are currently the method of choice. As I discussed in a previous blog (“WES, an alternative to ELISA”, 4/15/15), ELISAs possess the sensitivity and specificity required for GMOs, but not the day-to-day precision and are labor intensive. Automated Western analysis, as performed with the Wes™ system (Protein Simple®), alleviates many of the issues of with ELISA. It shows excellent day-to-day precision and is highly automated. However, both of these methods have relatively low sample throughput, 25-35 samples per plate with total analysis time from 2.5 – 18 hours. Given the hundreds of samples generated for a single GMO field trial, each of which require analysis of 10-20 different proteins, these techniques require man-months of analysis time. While ELISA can be multiplexed, this too is a laborious process and is better suited for analysis of a single matrix (plasma).

Within the last few years the use of proteomics, specifically LC/MRM/MS, has appeared in the literature for the analysis of GMO proteins. This technique appears to possess all the requirements necessary including high throughput. To provide the specificity required the mass/charge ratio must be determined for each of the proteins of interest. Typically the necessary sensitivity cannot be obtained analyzing intact proteins; therefore, proteolytic peptides are utilized. The entire plant extract is proteolytically digested (e.g. trypsin, Lys-C, etc.). This mixture of thousands of peptides is then analyzed by UPLC/MS/MS. Most of the peptides co-elute with multiple other peptides, but through the use of powerful proteomic software the peptides of interest can be teased out of the background. The chromatographic resolution is then optimized and the use of MRM (Multi Reaction Monitoring) is incorporated. In MRM, the peptide ion is separated by its mass/charge ratio in Q1, reacted in Q2 to produce daughter ions fragments which are further separated in Q3. MRM increases the sensitivity of the method 100-fold and provides an additional level of specificity by monitoring two daughter ions. Optimal precision is obtained through use of internal standards, usually synthetic ¹³C-labeled daughter ions. While this analysis requires substantial development time (as does ELISA), once developed it is a highly automated (walk-away). Its ultimate advantage is that MS/MRM can be multiplexed; there are reports of twelve or more proteins quantitated in a single analysis. This results in sample throughput 5-10 fold greater than ELISA or Western.

In summary, MRM/MS has the following advantages:

  • Accurate

  • Highly specific – two daughter ions

  • No requirement for antibodies

  • High throughput/multiplexing

  • Highly automated

While currently not the method of choice, MRM/MS seems poised to make enormous inroads for the analysis of protein levels in GMO plants.