Metabolomics

Weighty science

Metabolomics center delves into the unknown

Emily Kummerfeld | Bond LSC

What do you do when you have an unknown substance and need to know what it’s made of? Or what if you know what’s in it, just not how much?

Scientists turn to metabolomics to figure out what these pieces are.

Lloyd W. Sumner, Director of the Metabolomics Center at MU’s Bond Life Sciences Center, said analyzing a sample is like going to the doctor and having blood drawn to assess what’s happening inside of your body.

“Plants can’t tell us what’s going on, animals can’t do that either, so we need high resolution biochemical phenotyping to understand how organisms respond to stress and disease. Instead of profiling one, five, 10 or 30 compounds, we’re profiling hundreds to thousands of metabolites, and we use that to assess the biochemical phenotype and how the system is responding,” Dr. Sumner explained.

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Lloyd Sumner, biochemistry professor and Director of the Metabolomics Center at Bond LSC. | photo by Morgan McOlash, Bond LSC

This is achieved using methods called chromatography and mass spectrometry. These methods separate and analyze hundreds to thousands of metabolites to better understand the biochemistry of living organisms. Metabolites are the molecules a cell creates that provide the building blocks and energy sources enabling a plant or animal to grow, reproduce and respond to its environment. These small molecules can tell us a lot about an organism.

So how exactly how does mass spectrometry work? It really is the science of weighing molecules. It can identify small molecules, quantify known compounds and reveal structural and chemical properties. Crime shows on TV employ the technique in forensic investigations to analyze the molecular composition of unknown substances found at a crime scene.

Let’s break down the process for you.

The first step is actually chromatography, where the molecules in a mixture are separated based on chemical properties. This helps to see individual metabolites within complex mixtures from plants, animals and microbes. Two of the most common types are gas and liquid chromatography. Sumner’s lab uses both since some molecules cannot be heated during gas chromatography and need to be separated via liquid chromatography.

In gas chromatography, a sample is heated until it vaporizes and then travels through a thin glass tube and interacts with a coating on the tube wall. These interactions vary and help separate all the sample’s parts. The speed at which each separate molecule travels helps us determine the identity of each part.

Here’s where the mass spectrometer takes over. As each small molecule/metabolite exits the chromatography column, it enters through the inlet system, and passes through an ionization system that puts a charge on the molecules, since a mass spectrometer does not weigh actual mass but rather the mass-to-charge ratio of the molecular ions. Once the ions are formed, they go through the mass analyzer, which will tell the ions apart based on their mass to charge ratio. From there, the ions hit a detector where a resultant current is measured. This gives scientists a graph of sorts, showing a series of peaks for each substance, with higher peaks indicating more of the molecule in a sample.

This data from both chromatography and the mass spectrometer is then used to figure out exactly what was in the unknown sample.

Control is the name of the game when it comes to getting accurate results. The system keeps samples in a vacuum so molecules can move around without colliding with other gas molecules, because bad things can happen when they do.

“They can deflect the trajectory of the ions as they’re moving through the instrument, there can be charge loss and neutralization of that, but ultimately all of those affect sensitivity,” Sumner said.

Depending on what Sumner’s team is trying to figure out, other types of mass analyzers can come into play. From quadrapole mass analyzers to magnetic sector instruments, to time-of-flight analyzers, they separate the ions in different ways.

Not all mass analyzers are equal as Dr. Sumner explained, “these different types of mass spectrometers have different performance metrics. And as resolution and sensitivity go up, usually cost does, too. But for the most part, we use mostly TOF (time-of-flight) analyzers because they have good mass resolution and the cost is kind of modest.”

The way a time-of-flight analyzer separates ions is actually quite simple. A burst of ions is emitted and accelerated, and the ions are measured based on their flight time over a specific distance, meaning the smaller and lighter the ion, the faster it will be, with each molecule having a unique time-of-flight.

So how does Dr. Sumner use mass spectrometry in his own research? Currently, his work focuses on plant biochemistry.

“We’re trying to understand how plants synthesize triterpenoids. These are important plant compounds that plants use them to defend themselves. They can’t get up and run away, so they make these defense-related compounds,” Sumner said.

The Metabolomics Center benefits more researchers than just Sumner’s team.

For example, his center is collaborating with other faculty on an animal stress test for milk. That’s because the technology at the center can be applied widely to science not only here on campus but nationally and internationally.

An added bonus for Sumner is that his center feeds his fascination with sophisticated analytical instrumentation and electronics, or “shiny toys,” since once “you understand how they work, you get a greater appreciation for them,” he says.

Lloyd Sumner is a professor of biochemistry and director of the University of Missouri Metabolomics Center at the Bond Life Sciences Center. The Metabolomics Center, which opened in August of 2016, is one of ten research core facilities at the University of Missouri. Learn more about the center at http://metabolomics.missouri.edu.

 

Research Core offers new capabilities

Grand opening highlights specialty of large-scale metabolite profiling
By Phillip Sitter | Bond LSC

Dr. Zhentian Lei , assistant director and assistant research professor of the MU Metabolomics Center, provides an overview of an ultra high-pressure liquid chromatograph coupled to mass spectrometry for the large-scale profiling of metabolites at the University of Missouri Metabolomics Center open house on Aug. 12. | photo by Zivile Raskauskaite, Bond LSC

Dr. Zhentian Lei , assistant director and assistant research professor of the MU Metabolomics Center, provides an overview of an ultra high-pressure liquid chromatograph coupled to mass spectrometry for the large-scale profiling of metabolites at the University of Missouri Metabolomics Center open house on Aug. 12. | photo by Zivile Raskauskaite, Bond LSC

You might think you’ve entered the inside of a pinball machine for a moment when you enter lab 243 at the Bond Life Sciences Center.

But the wires and tubes strung around the room, connected to large instruments that produce sounds of whirring fans, humming motors and hissing pumps, are just part of the University of Missouri’s newest core facility, the MU Metabolomics Center.

At its grand opening and open house Friday, August 12, there was even a counter-top half-pipe with metal ball bearings to shoot down it as a demonstration of time of flight mass spectrometry.

This new center will serve as home of high-tech chemical analysis services that scientists in Bond LSC, across campus and the country can use to better understand the organisms they work with on a molecular level.

Lloyd Sumner, director of the MU Metabolomics Center, and Assistant Professor Ruthie Angelovici discuss the use of NMR for metabolite identification during the University of Missouri Metabolomics Center open house on Aug. 12. | photo by Zivile Raskauskaite, Bond LSC

Lloyd Sumner, director of the MU Metabolomics Center, and Assistant Professor Ruthie Angelovici discuss the use of NMR for metabolite identification during the University of Missouri Metabolomics Center open house on Aug. 12. | photo by Zivile Raskauskaite, Bond LSC

“We have a series of experiments that allow us to profile hundreds to thousands of different metabolites, and that gives people a large-scale, high resolution biochemical traits for whatever they’re looking at, whether it be plants, microbes or animals,” explained Lloyd Sumner, director of the center. “That is useful in understanding what is happening in response to stresses, disease, drug treatment or pest/pathogen interactions that occur in nature.”

Metabolites are the building blocks and energy sources that fuel your metabolism. In your body, what you eat and drink is processed and yields small molecules that are ready to become raw chemical material for construction processes and energy to fuel these processes, like energy stored in the form of fats and lipids, amino acids for the construction of proteins and enzymes. Metabolite are essentially the raw materials.

In order to be studied, complex metabolite mixtures are separated and observed as individual, uniquely identifiable molecules.

This separation can be accomplished in a couple different ways.

“We have instruments that couple chromatography with mass spectrometry. We use that for comparative profiling. Some of the instruments utilize gas chromatography, some of the instruments use liquid chromatography. Chromatography is the technology used to separate these complex mixtures into its individual components. Once we have the mixture’s components separated, we weigh them and that gives us an idea of their identification,” Sumner explained.

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These internal components of a triple quadrupole mass spectrometry are used for explaining how the instrument helps identify the metabolites within a sample during the University of Missouri Metabolomics Center open house on Aug. 12. | photo by Zivile Raskauskaite, Bond LSC

Mass spectrometry works by bombarding molecules with electrons. This bombardment process generates charged molecules that can also fragment into smaller, electrically-charged pieces. These charged pieces can then be “weighed,” or separated, according to their mass-to-charge ratio and identified.

“Something that we find a lot of the time is that we see metabolic differences, but we can’t always identify all of the metabolites associated with those differences. In those cases, we also use the gold standard for chemical identification of unknown molecules,” Sumner said of the nuclear magnetic resonance (NMR) spectrometer in the corner of the lab.

A person points at a 600 MHz Nuclear Magnetic Resonance Spectrometer used for metabolite identification during the University of Missouri Metabolomics Center open house on Aug. 12. | photo by Zivile Raskauskaite, Bond LSC

A person points at a 600 MHz Nuclear Magnetic Resonance Spectrometer used for metabolite identification during the University of Missouri Metabolomics Center open house on Aug. 12. | photo by Zivile Raskauskaite, Bond LSC

Placards warn people that when NMR produces a magnetic field 235,000 times stronger than the Earth’s — by comparison, a typical refrigerator magnet’s field is about 83 times as strong as the Earth’s.

Sumner explained that most people at Bond LSC won’t use the equipment directly themselves. The center’s Assistant Director Dr. Zhentian Lei and other staff will perform most analyses and training users to prepare, process and understand their data.

Sumner said “we train our core users to do their own sample preparation, data processing and data interpretation. Most of the equipment we have in here [cost] hundreds of thousands of dollars, and so we actually have staff that will do the data acquisition, and we try to make it more cost-effective for users by training them to prep their own samples and process their own data.”

The training workshop in metabolomics will be August 15 through 19. The training Monday through Thursday will be hands-on, and Friday will be a symposium day highlighting current metabolomics research. We will likely offer another training workshop in the Spring of 2017, and then annually thereafter.

For more information on using the MU Metabolomics Core or future training, email Director Lloyd Sumner at sumnerlw@missouri.edu or Assistant Director Zhentian Lei at leiz@missouri.edu.