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.
