The samples shipped to the mass spectrometry lab at EMSL bear labels from all over the world, from bacteria grown in U.S. laboratories to aerosol particles trapped in the Amazon basin. Although the contents vary greatly, they share two things: a complex molecular makeup and a place in the starting lineup of samples waiting for a spin in the new 21 Tesla Fourier transform ion cyclotron resonance, or 21T FTICR, mass spectrometer that was designed and built for research at EMSL.
Ten years ago, the studies of these samples wouldn’t have been possible. That’s when a group of 100 scientists from 40 institutions visited EMSL to discuss advancements in measurement science that would be needed in the following decades. One of the priorities identified was development of a 21T FTICR mass spectrometer. Subsequent meetings around the world confirmed the need for this kind of capability to enable “breakthrough discoveries in the understanding of complex systems,” according to a 2008 workshop report on this subject. Scientists saw the potential for addressing what they called “analytical grand challenges” such as studying biochemical pathways, cellular communication and microbial communities at the molecular level.
“We recognized the opportunity to advance our understanding of complex systems across a broad spectrum of science, from characterizing organic aerosols to understanding microbial community dynamics to achieving high-resolution chemical imaging,” says David Koppenaal, EMSL’s chief technology officer and one of the leaders of a 2008 workshop focused on identifying the science challenges for such a next-generation mass spectrometer.
These scientific questions drove development of the new 21T FTICR mass spectrometer, which is now available to scientists around the world for their research.
“We’re poised to realize the possibilities of scientific studies we only imagined 10 years ago,” says Koppenaal.
Now, scientists will be able to obtain molecular-level information to improve biofuel production efforts as well as our understanding of climate effects on carbon cycling in the soil and carbon transformations in the atmosphere.
“Scientists will be able to pinpoint the molecular makeup of complex chemical systems with near unequivocal certainty,” says Koppenaal. “This instrument provides data with mass accuracy to within six decimal points. Conventional instrumentation today doesn’t have that kind of resolution or mass accuracy.”
The system is expected to be fully operational by October. A Special Science Call went out last year for studies that incorporated EMSL’s recently developed technical resources, including this new mass spectrometer. Researchers are keen to get results with the six inaugural studies selected through the call.
Center of Attraction
Lili Paša-Tolić, EMSL’s lead scientist for mass spectrometry, said the “21T” has significantly ramped-up performance in terms of mass measurement accuracy and mass resolving capabilities. “At higher magnetic fields the accuracy, mass resolving power, sensitivity and dynamic range improve either linearly or quadratically.”
Through a top down proteomics approach advanced by Paša-Tolić, the enhanced resolving power of the 21T will allow proteins up to 150 kDa (daltons) to be parsed in their entirety. With current resolution, researchers typically need to first pick apart big proteins and analyze them bit by bit – often missing key information in the process.
This top-down method is of particular interest to Jonathan Walton, a plant biologist at the Michigan State University-Department of Energy Plant Research Laboratory and member of the DOE Great Lakes Bioenergy Research Center, which is funding his study. His work with fungi that break down plant walls led him to study cellobiohydrolases, or CBH1. This group of enzymes is adept at degrading the tough cell walls of lignocellulose, an abundant biomass from which we could produce biofuels if the process operated more efficiently and economically.
“These enzymes are particularly tricky to work with,” says Walton. To express the cellobiohydrolases, he inserts their genome sequence into yeast that is more tractable than the enzyme’s native fungal source. But Walton found that yeast alter CBH enzymes by adding sugars to them after they’re biosynthesized. This “post-translational glycosylation” can change enzyme function.
With the 21T, Walton hopes to see precisely what’s happening, and then correlate the glycosylation modifications to key enzyme properties such as thermostability and activity.
“From that we’ll be able to design better enzymes,” he says.
Although he’s a first-time EMSL user, Walton plans to follow this work with more traditional “bottom up” proteomics and glycomics using the facility’s mass spectrometry and nuclear magnetic resonance spectrometers to understand in greater detail how glycosylation occurs. And he’s already submitted another proposal to look at another group of cellulases that degrade lignin.
“A lot comes down to understanding what happens when you express proteins from one organism into another,” Walton says. “Strange things happen, and the new mass spectrometer at EMSL is the first step to understanding what’s going on.”
Another researcher working on the lignocellulose puzzle is Blake Simmons, the chief science and technology officer and vice president of the Deconstruction Division at the DOE Joint BioEnergy Institute, based in the San Francisco Bay area.
Simmons refers to lignin, one of the primary components in lignocellulosic biomass, as “the Gordian knot” of biofuels. To start cutting that knot, he hopes the 21T will help him map the metabolic pathways of organisms that break down lignin. Then he’ll chart the way metabolism changes with varied environments, growth substrates and end products.
“Once we build that map, we can translate that knowledge into biorefining, and then deploy it to convert not only sugars to fuels, but also from lignin to fuels and chemicals,” he says. His studies are funded by DOE’s Office of Biological and Environmental Research.
Right now, Simmons and his team are working with Klebsiella and Enterobacter, two organisms he calls the “lab rats” of the bacterial world. During soil studies in the cloud forest of Puerto Rico, scientists discovered that both of these bacteria can degrade lignin, but there are differences in how the two use carbon and sugar resources. For his 21T samples, Simmons’ team tracked the bacteria as they were grown in the lab and fed specific substrates, one at a time. At pre-set intervals, his team harvested the cells for 21T analysis of the cell fractions – the lipids, proteins, enzymes, metabolites and so on – to identify the molecules present during metabolic changes.
“We’re trying to create an electrical circuit equivalent of metabolism in these organisms: finding out where those fluxes are turned on, turned off, held constant,” says Simmons. “Once we get that electrical wiring diagram of metabolism then we can start doing what we do best: fiddling with it, engineering it, and optimizing it for our specific purpose.”
Another set of samples headed for the 21T also started with a study in Puerto Rico’s Luquillo Experimental Forest. There, microbiologist Jennifer Pett-Ridge, the lead scientist of Lawrence Livermore National Laboratory‘s Genomic Science Biofuels Scientific Focus Area, is using soil samples from various mountain elevations to study how climate change will affect the conversion of soil carbon into highly mobile dissolved organic matter.
Tropical forest soils are thought to store a huge amount of carbon, but changes in global weather patterns may make these terrestrial carbon dioxide sinks less reliable, notes Pett-Ridge. The tropics are expected to experience longer droughts punctuated by intensive rainfall, so they may become less consistently wet. Under those climate conditions, the carbon adsorbed onto soil mineral surfaces is dramatically affected when the soil goes from oxygenated to an anaerobic state.
“Carbon goes from tightly bound to mineral surfaces, to more amorphous loose association; this will affect the chemistry and quantity of dissolved organic carbon,” says Pett-Ridge. Her studies are supported by a DOE Early Career Research Program award through the Office of Biological and Environmental Research.
With soil samples from forest sites that range from wet tropical to the colder cloud forest, Pett-Ridge hopes the 21T will help her understand how changes in soil oxygen and redox – “whether there are many, many changes or slow redox every week to two weeks”– affect the total amount of carbon released into the dissolved pool.
“I also want to know about the quality – the molecular composition – of the dissolved pool,” says Pett-Ridge. “That’s where the new mass spectrometer comes in.”
Right now, she can detect elements in soil samples at the single element/isotope level with the NanoSIMS (nanoscale secondary ion mass spectrometry) at EMSL. If the 21T performs as anticipated, Pett-Ridge could identify molecular-level components in her samples, such as lipids, proteins and peptides. She and collaborator Aaron Thompson, a biogeochemist at The University of Georgia, started a pilot study on soil samples with the 15T mass spectrometer in use at EMSL now. Next, if the new instrument proves to be the right tool for her research questions, she hopes to expand her work with samples from multiple lab incubations of tropical soils.
“The long-range goal is to say something more globally about tropical soils and how their microbial communities and their carbon chemistry are responding to climate change,” says Pett-Ridge.
The 21T will also play a role in better understanding carbon chemistry changes in the atmosphere. Soon, the instrument should produce data sets with details on the composition of samples of aerosol particles collected by EMSL researcher Alexander Laskin from pristine Amazon areas, and from areas directly affected by emissions from Manaus, a city in Brazil. A team of aerosol scientists, including Laskin, Sergey Nizkorodov, a researcher at the University of California at Irvine, and their co-workers are anxiously awaiting the launch of the 21T instrument.
“The goal is to learn how natural aerosols are affected by emissions from a major city,” says Nizkorodov. His studies are funded by the National Science Foundation and National Oceanic and Atmospheric Administration.
In areas of the Amazon with little manmade pollution, biogenic emissions get oxidized and become complex aerosol mixtures made primarily of carbon, oxygen, hydrogen and some nitrogen. With current tools, scientists have been able to sort through the composition of these aerosols. However, near a city, anthropogenic effects add more nitrogen, sulfur, silicon and other elements into the atmospheric mix.
“Under those conditions, there are so many possible chemicals that, right now, no existing mass spectrometer can resolve all of them,” says Nizkorodov. “This 21T has the capability of unambiguously resolving this very complicated mixture and teaching us something new about the atmospheric chemistry of aerosols.”
With new information from the 21T, Nizkorodov hopes to learn whether mixing emissions from the city with those from the forest generates unique compounds. If there are new compounds, the next step will be determining what special properties they have: Do they absorb white light? Do they have toxic consequences?
Nizkorodov also plans to run model aerosol samples from a smog chamber. He’s specifically interested in the effect of humidity on aerosol samples because most prior experiments were performed in dry chambers. With the information currently available, it’s difficult to predict whether the humid environment in the Amazon will alter the predicted chemical transformations of aerosols. So, he plans targeted experiments in which he’ll introduce nitric oxide, sulfur dioxide and dry air into a chamber loaded with a mixture of volatile organic compounds; then he’ll use exactly the same mix in humid air and see whether this creates any difference at the molecular level, and on the properties of the aerosols.
“The 21T will definitely take our studies to the next level,” says Nizkorodov. “We’ll be able to understand the aerosol chemistry so much better. I’m very excited to be one of the first users.”
The new mass spectrometer is the single biggest investment that EMSL has made in an experimental instrument, notes Koppenaal.
“This instrument will really open the door to a whole new area of scientific inquiry,” says Koppenaal. “We’re on the forefront of better understanding molecular-level science in the natural environment.”
Elizabeth Devitt is a science journalist and freelance writer.