Robert "Bob" Hettich has spent much of his career at the Department of Energy's Oak Ridge National Laboratory demonstrating how high-performance analytical measurements, in particular mass spectrometry, can yield remarkable insights into the mysteries of tiny microbes and their impact on larger systems such as plants and humans.
He uses mass spectrometry, a technology that measures the mass/charge of ions generated from molecules, to explore cellular machinery and processes that reveal how bacteria, fungi and viruses associate and interact with each other and their hosts. His research on biomolecules from complex environments has enabled fundamental insights into how these microbes function and adapt in microbiomes. This information helps guide numerous applications, such as how microbial consortia - teams of microbes that work together - enhance plant health and can be used to support the domestic bioeconomy.
Hettich is an ORNL Corporate Fellow and leads the lab's Bioanalytical Mass Spectrometry group. He is widely recognized for his mass spec expertise, and is a founder and global leader in the field of metaproteomics , the identification and analysis of the proteins and peptides produced by microbial communities.
Hettich collaborates with numerous researchers at ORNL and other institutions to characterize how microbes interact with each other, influence soil nutrient flow, affect bioremediation, aid the production of biofuels and bioproducts, help plant hosts adapt to conditions such as drought, nutrient stress, disease and pests, and how they can influence or respond to human health conditions. He has also studied higher order protein structure, exploring the three-dimensional shape and folding of proteins that determine how they function and interact with other molecules, to better understand biological processes.
Hettich's work is supported by a variety of DOE Office of Science Biological and Environmental Research program projects, including the ORNL-led Center for Bioenergy Innovation , or CBI, and the Plant-Microbe Interfaces Science Focus Area , or PMI SFA.
Across the span of his 39-year career, Hettich has been involved in the development and demonstration of several generations of biological mass spectrometry technologies at ORNL. He is currently overseeing the establishment of new cutting-edge mass spec equipment that can significantly accelerate ORNL discoveries for better plants and microbes.
Q: What are the new mass spec capabilities at ORNL?
We've just installed two new high-performance mass spectrometers at ORNL, which should provide a big leap forward in our capabilities for faster scanning speed, higher resolution and greater sample throughput.
Our current mass spec instruments scan at a frequency of 8 Hertz, generating eight mass spectra per second. The primary new mass spec instrument operates at 200 Hz, yielding 200 mass spectra per second, for a 2500% increase over our existing equipment. Additionally, this instrument provides ultra-high mass resolution of 480,000, allowing for incredibly precise measurements of complex molecules like proteins and metabolites. A second new mass spec instrument we've acquired can scan up to 40 Hz, enabling faster speeds over current instruments while providing high mass measurement capabilities for interrogating intact proteins and other biomolecules.
We also have new computing capabilities directly interfaced to these mass systems to handle the influx of data from these significant upgrades, providing real-time data collection, analysis and storage. As our datasets expand, they will continue to fuel ORNL's artificial intelligence and high-performing computing capabilities to further expand our knowledge of complex biological systems.
Because we are one of the few groups internationally recognized for driving and developing metaproteomics - the large-scale study of proteins produced by entire microbial communities - part of my job is to keep the technology toolset moving forward. We want to continue expanding our ability to make the best measurements possible and thereby open new doors of understanding that have not been previously possible.
Only a few of these instruments are in use around the world, and most are focused on human health research. The combination of our ORNL science missions and expertise in plant and microbial biology and these new instruments makes us very unique among research institutions. These capabilities reinforce our standing as a top-tier institution for metaproteomics, boosting our innovations for advanced fuels, chemicals and materials, and natural ecosystem resilience.
Q: What advantages can we expect from the instruments?
We use liquid chromatography-tandem mass spectrometry, or LC-MS/MS, in which samples are first injected into an LC column and compounds are separated based on polarity, size or other chemical properties. As they exit the column, they then enter the mass spectrometer, where they're ionized, fragmented and detected based on their mass-to-charge ratio. Computational tools are then used to match those masses to known predicted molecules from the DNA sequences, identifying precisely what's in the sample, how much of it is there and even its sequence.
The samples we're analyzing are very complex mixtures, and thus the mass spec is trying to scan as fast as it can to record all of the information coming off the column in real-time. With the older instruments and their slower scanning speeds, we were missing at least one-half of what's coming in off the column. Obviously the newer faster scanning instruments provide much more expanded detection depth as well as better measurement quality, enabling a deeper and more comprehensive view into the molecular machinery of these complex biological systems. There is also a substantial gain in measurement time: our current measurements require about 3 to 5 hours per sample on the current MS equipment; this can be reduced to about 30-60 minutes on the newer systems. This means faster results and much higher throughput.
With the 480,000 resolving power on the new primary instrument, we gain the ability to differentiate peaks sitting very close together in the mass spectra. For example, stable isotope labeling is a common experiment to trace the flow of carbon or nitrogen through a biological system. With this high resolution, we should be able to more sensitively and accurately characterize a range of stable isotopic labels, which will allow us to better track, for instance, nitrogen limitation as a function of carbon flow. Such knowledge can help us understand how carbon flow affects nitrogen uptake and growth in plants.
Another example is the ability to identify microbes that we might not have seen before in the soil environment around plants or on plant leaves just because their abundance was so low. The new instrumentation provides significant increases in measurement sensitivity (at least 5X or so) and thus will provide a much richer view. We should now be able to see microbes and microbial functions that we didn't know were present and important simply because we didn't previously detect them.
Q: What have we accomplished so far with the new technology?
We're already testing increased speed, sensitivity and the coverage of protein profiles. Analysis of a complex microbial mixture revealed more than 250,000 unique peptides measured in a 60-minute session, almost 10 times greater measurement depth that what we could achieve with existing equipment and longer analysis times. Additionally, we evaluated a soil sample for depth of measurement performance and the ability to measure microbial proteins. The resulting protein numbers were very strong, again vastly superior to existing methods.
Q: What new science can the instruments enable?
ORNL has extensive experience and success in using genome-wide association studies, or GWAS, as part of the CBI project to link genetic variants to traits in plants. A complementary approach would be to link actual genetic function, i.e. proteins, to favorable traits - suggesting that proteome-wide association studies, or PWAS, could be valuable. However, the ability to do PWAS has been unachievable due to technical limitations of measuring large sample sets.
With a PWAS, you learn what the proteins and metabolites are actually doing, rather than their genomic potential that is revealed in the genome. Simply put, the genome provides the cookbook while the proteome/metabolome reveals the cake. By monitoring proteins across thousands of samples, you can link actual function to a gene more accurately.
By combining GWAS and PWAS, we should be able to more comprehensively identify the molecular mechanisms driving plant response to the environment - factors like drought and disease resistance, greater biomass and crop yield. This would reveal gene-protein associations related to metabolic pathways, helping us boost the production efficiency of new bio-based fuels, chemicals and materials, and identifying the best microbial pathways to break down and recycle plastics, for instance.
For the PMI SFA project, the new instruments can help us better understand the microbial communities that live in plant roots, including how those microbes communicate with plants, the exchange of small molecules, what controls the growth of the microbiome, and whether there are carbon or nitrogen limitations.
The new MS technology also gives us the ability to analyze intact proteins. Rather than digesting proteins down to peptides and looking at the building blocks, we can now perform top-down analysis of intact structures. We can monitor things such as protein interactions or how proteins perform when they are modified with chemical tags such as methyl and phosphate, and how protein modification can dynamically control function.
Q: What other science missions can these capabilities support?
In addition to our focus on environmental microbiology, we have also been very active in human microbiome research. These systems are similar to the plant microbiome - they both contain a complex eukaryotic host that contains an established microbiome, which can differ under healthy vs. unhealthy conditions.
We can use what we know about plants and microbial interactions to develop a common architecture to analyze complex environments in the human body. We're currently analyzing fecal samples for a project looking at the connections between the human microbiome in the context of Parkinson's disease and rheumatoid arthritis. Even with initial testing, we're seeing about 10 times deeper measurement results with the new mass spec. Our new high-throughput capabilities should provide a much enhanced view of what constitutes a healthy human microbiome compared to one that's linked to disorder or disease.
Q: What do we hope to achieve, ultimately, with the new technology?
With our new mass spec capabilities, we gain several important advantages: resolution, or how sharp the peaks are that will aid in precisely identifying molecules; scan speed, which increases the number of samples we can run in a given time; and much greater mass accuracy and sensitivity. It is quite likely that these new performance metrics will not only better address current science questions but also expand the field of view in new areas.
Together with other capabilities at ORNL such as neutron science, high-performance computing and artificial intelligence, cryo-electron microscopy, X-ray crystallography and automated, multimodal phenotyping, we will have the power to open doors to new layers of research, making what has been untouchable now tractable. Ultimately these new instruments advance our ability to untangle and understand complex living systems holistically rather than as individually operating functions.
UT-Battelle manages ORNL for DOE's Office of Science, the single largest supporter of basic research in the physical sciences in the United States. The Office of Science is working to address some of the most pressing challenges of our time. For more information, please visit energy.gov/science . - Stephanie Seay
