The Real CSI
Mass spectrometry has managed to creep into the mainstream consciousness - albeit without most people realizing it's even there. Popular television shows like CSI have proliferated images of investigators collecting samples from crime scenes and then "analyzing them in the laboratory" to understand their chemical makeup. That's analytical chemistry and mass spectrometry. That's Lloyd Sumner's world - without the crime, of course.
Sumner, a chemist by training, leads the Noble Research Institute's mass spectrometry laboratory. His nine-member team uses some of the scientific community's most advanced equipment to conduct mass spectrometry, which literally revolves around sorting and weighing individual atoms and molecules. It's a detailed and painstaking process conducted on a level so small it defies most people's imaginations.
"For example, a molecule of sucrose (table sugar), a common molecule found in plants, is about one nanometer in size, which is roughly one millionth the size of a grain of sand," Sumner explained. "A similar comparison to help put this into perspective would be 1 foot relative to 189 miles."
Plants make a variety of complex molecules known as natural compounds, many of which have yet to be characterized. The weight of a molecule seems insignificant, but in reality the weight can reveal the molecule's chemical identity. Once a scientist understands the chemical makeup of a compound, he can begin to search for the mechanisms and genes within the plant that produce it. "We're detectives," Sumner said. "We're literally figuring out the functions of genes in plant chemistry, which is not a trivial task."
While many of the Noble Research Institute's researchers study the genes directly, Sumner's group moves to the other end of the spectrum and works backwards by conducting large-scale biochemical analyses of proteins and metabolites.
"The cellular machinery can be envisioned as a construction site. DNA or the genetic code serves as the blueprint from which work orders are issued as messenger RNA," Sumner said. "Messenger RNA is used to build proteins, which serve as the construction workers of life. The proteins then build cells and tissues using metabolites, which are like bricks and mortar."
Primary metabolites serve as building blocks (amino acids) or as energy sources (sugars and fats). Secondary metabolites, the focus of Sumner's research, function as unique communication signals during plant interactions with the environment. They also act as chemical warfare agents involved in plant defense since plants cannot run from their enemies.
To a greater extent, large-scale profiling of hundreds to thousands of metabolites, known as metabolomics, offers a definitive view of the "metabolic status" of an organism. "It's a high resolution biochemical fingerprint, or a snapshot, of a cell's biology," Sumner explained.
In essence, scientists study the metabolite and protein populations in a control plant and then compare their findings to a plant that has been purposely stressed, such as with drought or disease. They then focus in on the differences (increases or decreases) of particular metabolites. Understanding these chemical changes becomes the basis of identifying the metabolites, proteins and gene(s) that are responsible for such changes.
These changes can only be detected using specialized instrumentation. The nerve center of the facility is a series of bays that contain nine mass spectrometers, each with its own abilities and characteristics. These various mass spectrometers differ in both sensitivity and how they receive samples - as a solid, liquid or gas.
There are an estimated 10,000 unique metabolites in the plants being studied at the Noble Research Institute. Using the various mass spectrometers, the group can detect more than 1,000 of these. "It's very much a divide and conquer situation," he said. "Each molecule has different requirements, and we match them to the appropriate instruments. The mass spectrometers are just fancy tackle boxes. As the technology improves, we'll get even better at catching more trophy fish."
For the scientists, the technology remains both the gateway and the barrier to delving deeper into metabolomics and proteomics (similarly, the large-scale analysis of proteins). For the layman, the technology's power seems almost incomprehensible. For instance, when discussing the sensitivity of the instruments, Sumner said, "The sensitivity of a mass spectrometer is one part in a quadrillion which is similar to being able to differentiate one hair from all the hairs on all the cows in Oklahoma."
While the advanced technology makes the job possible, it doesn't make it easy.
Television often highlights the equipment and the processes used during mass spectrometry. What's unfortunately misrepresented is the speed of the process. The results usually zip back to the TV detectives in minutes. That's not the case for real scientists.
David Huhman, analytical chemistry core facility coordinator, explained that samples can take three or more days just to prepare. Each sample will typically take an hour or two to analyze, meaning an experiment with several hundred samples can require days, weeks or months of continuous data acquisition. Working with plants doesn't help the timeline either. Eight to 12 weeks for growing the plant materials usually precedes the sampling process.
Raw data must then be processed by computers (at least a full night) and analyzed by the staff, which might take days or weeks. "You must be meticulous in your preparation," he said. "If you make a mistake, you may not see it for two or three weeks. And if you do make one, it can cost you weeks of effort."
The work may be time consuming, but studying metabolites has brought about key findings that may impact agriculture in the future.
While studying the impact of plant stress on secondary metabolites, the Sumner team unearthed an unexpected compound identified as hispidol, which had not previously been linked to stress responses. "Because we were performing integrated RNA profiling, we could simultaneously identify two genes correlated with the biosynthesis of hispidol," Sumner said. "These discoveries can now be used to engineer plants to produce hispidol to fight against fungal pathogens and disease."
Another success for the laboratory revolves around cotton root rot, a disease that devastates more than 2,000 plant species throughout the southwestern United States. Wensheng Li, Ph.D., from the Sumner group, discovered that plants like alfalfa - an agriculturally and economically important legume - have a series of chemical responses that can defend the plant against fungal infection, but that these responses are not initiated soon enough to combat the attack.
The group is now trying to tweak the plant's response and engineer plants like alfalfa to initiate the secondary responses earlier to combat cotton root rot.
Sumner's group is seeking multiple viable solutions to protect crops like alfalfa from cotton root rot.
Working with his longtime mentor, Rick Dixon, Senior Vice President and Plant Biology Division Director at the Noble Research Institute, the Sumner group sought to understand how white lupin is able to successfully combat cotton root rot. The researchers theorized that particular chemical compounds produced by lupin roots were responsible for its defense against the disease. Similiar chemicals are found in large quantities in the fruits of a local tree, the Osage orange.
The Sumner/Dixon groups were correct in their theory. They subsequently identified the chemical compound wighteone, present in both lupine and Osage orange, as a potent inhibitor of the growth of cotton root rot fungus. Wighteone possesses antimicrobial properties and seems directly linked to the Osage orange's hardiness. Although alfalfa does not naturally produce wighteone, it does produce highly similar compounds. The groups are now seeking the specific genes that would enable the one-step biosynthesis of wighteone in alfalfa.
"It's these discovery events that open new possibilities for metabolic engineering and the improvement of agriculture," Sumner said. "The challenges facing agriculture in future generations are going to be immense. We are utilizing highly sophisticated instrumentation to enable vital discoveries and enhance our understanding of plant biochemistry."