Mass Spectrometry is Advancing Single Cell Biology Research

The Sweedler group at the University of Illinois at Urbana Champaign (UIUC) are at the forefront of analytical discovery, thanks to Bruker’s range of high-spec mass spectrometry instruments.

Working with Bruker

Prof. Jonathan Sweedler’s research group at UIUC enjoys the advantages of an extensive range of analytical equipment, selected for its peak performance for his precise application needs.

 

For mass spectrometry imaging, Bruker’s technology leads the way. In particular, their MALDI imaging instruments have incredible figures of merit that other vendors don’t come close to.

The Department of Chemistry at UIUC

Since its founding in 1868, the Department of Chemistry has formed part of the University of Illinois at Urbana Champaign (UIUC) and supports a variety of fields of research, from chemical biology to analytical, inorganic, materials, organic, physical and theoretical chemistry.

UIUC is well-placed for ground-breaking chemical discovery, and has reliably retained a spot at the top of national chemistry department ranks.

Professor Jonathan V. Sweedler, who has been a faculty member of the department for over a quarter of a century, carries out bioanalytical chemistry research at the juncture of analytical chemistry, bioengineering, neuroscience, and physiology.

Professor Sweedler, who boasts more than 365 peer-reviewed publications, is a frontrunner in the field. His team is presently made up of two research scientists, seven postdoctoral associates, 12 graduate students, two visiting academics and 11 undergraduate students.

Within their focus area of single cell biology, his group depends upon reliable instrumentation to take ever more precise measurements of minute biological samples, such as singular cells, in relation to a wide-ranging assortment of fundamental and applied analyses.

The Research

The Sweedler research group’s work can be roughly characterized as measurement science and technology, with an emphasis on the development of analytical techniques for the study of complex biological samples.

The Sweedler lab is well-known for developing, enhancing, and employing very sensitive approaches to examine a broad variety of model organisms, covering many phyla, with a primary goal of attaining a greater understanding of currently identified neurochemical pathways and discovering new and unusual pathways.

Sweedler is particularly fascinated with discovering and interpreting novel neuropeptides related to mechanisms of behavior, memory and learning.

Through the use of microscopy-guided MALDI (matrix-assisted laser desorption/ionization) mass spectrometry and capillary electrophoresis-coupled electrospray mass spectrometry, cells can be investigated for cell-to-cell signaling molecules to chart the chemical bases of connectivity within well-defined neuronal networks.

Using a combination of analytical approaches, the Sweedler group has chemically categorized thousands of neuropeptides over a broad range of animal models.

“It is a rather bold statement, but we have discovered and reported more novel brain peptides across the animal kingdom than any other group.This extends to discovering novel neuropeptide genes as well,” comments Professor Sweedler.

The study of single cell measurements is a field which is growing in importance. Through the development and use of sensitive and low sample-volume techniques, the genes expressed by an individual cell type can be explicated, and the dynamic phenotypes of apparently consistent cells under diverse external situations can be categorized and classified.

Professor Sweedler discusses the nature of his groups’ neurobiology research with mass spectrometry imaging:

We want to know what compounds are in the brain, where they are and when they were at particular location, as well as how they change based on behavior, learning and environment. Mass spectrometry shows you what compounds are present in the brain, for example, by removing a brain region and assaying it with liquid chromatography mass spectrometry.

We also want to know where the compounds are, and so we can take a tissue slice from the brain, conduct mass spectrometry imaging, and figure out the localization of different chemicals. For example, we can ask what happens in distinct brain regions of rats when you alter their diet, or induce learning with a learning task.

We perform the mass spectrometry imaging measurements with a Bruker ultrafleXtreme™ and our new solariX XR™. These instruments allow us to probe what’s in a particular location, for example within a layer of the hippocampus, and characterize the changes in neurotransmitters, neuropeptides, and lipids.

What the Group Works On

Single cell measurements are an exciting area. In the last few years, there has been an increase in interest and government funding channeled towards this technology, and therefore it is a hot area that scientists – undergraduates, graduate students and post docs – want to work in.

Because of this interest, around half of my group is now working in single cell biology, using some of the newest equipment we have, such as the solariX XR™.

Sweedler’s group continually aims to enhance the sensitivity and data content of their measurements, which is a goal well supported by access to the finest instrumentation. The solariX XR™ delivers this level of data through exceptionally high mass resolution detection.

The necessity of continual training is an issue found much more frequently in academia than in industry. Since the undergraduate, graduate and postdoctoral associates progress to new positions every few years, the degree of staff turnover is more rapid in academic environments than in industry.

While this does result in a limitation on the degree of expertise that can accumulate within the Sweedler group over time, it is a definite aid for the wider field. To illustrate, around 50 percent of Professor Sweedler’s students progress into the pharmaceutical industry, supporting a number of other groups with their knowledge.

According to Professor Sweedler, one advantage to this regular arrival of students and postdoctoral associates is that “new people tend to be fairly fearless and want to try new things.”

Although the turnover is regular, he has been lucky to have two senior scientists, Dr. Stanislav Rubakhin and Dr. Elena Romanova, who have remained with the team for approximately two decades.

One of our goals is to carry out complex measurements on cell-to-cell signaling, for example, on neurotransmitters and neuromodulators across the brain. It has been enabling to have these two expert senior scientists with broad expertise in sample preparation, LC mass spectrometry, mass spectrometry imaging, and a myriad of other related areas work on these projects.

They contribute to the long-term group knowledge of instrumentation and procedures and enable a number of projects to succeed.

Training

For brilliance in research carried out both within the group and in numerous collaborative projects, in addition to future contributions from group members who progress into science and industry, the degree of training delivered to students and staff in the group is paramount.

The requirements of the training can be varied, since members join the lab with differing levels of awareness and experience with mass spectrometry.

Single cell measurements with mass spectrometry require a steady hand, and potentially someone who doesn’t drink much coffee! We have found it best to train undergraduate students on equipment like the MALDI mass spectrometer where small samples can be used and the instrument is robust. For example, the ultrafleXtreme™ can handle a large number of sample plates, and so is great for training. If students want to progress to capillary electrophoresis - mass spectrometry, this makes a good transition for more experienced users as more attention to detail and greater potential for complications exist.

In addition to the receipt of training from senior staff and academics, Bruker offers numerous training events and ongoing support for the Sweedler group, allowing them to maintain a high level of knowledge on the most up-to-date equipment needed for pioneering research.

“Continuous training is one of the beneficial elements of collaborating with Bruker,” ... says Professor Sweedler, adding that “they supply an incredible amount of expertise – that’s worth a lot!”

This relationship is equally beneficial for Bruker; approximately 50% of the scientists that originate from the Sweedler lab go on to the pharmaceutical industry, carrying along their affinity for Bruker instruments.

A Challenge for the Group

It is evident that there are numerous difficulties to be addressed when undertaking research at the level of singular cells. A number of the issues that Professor Sweedler noted as needing to be overcome to achieve more robust single cell measurements were: more universal sampling protocols, increasing analyte detectability, and enhancing the throughput of mass spectrometry.

My group is continually fighting against detectability, attempting to achieve progressively lower detection limits. We have pushed creating better sampling approaches and we are using instruments where we can work at the single cell level and have the best detection limits – ... in practice this has always tended to be with Bruker, because of their increasingly sophisticated mass spectrometry technologies.

The group must also deal with a number of uncommon physical limitations. Professor Sweedler describes how his team dealt with one such encounter:

Studying brain chemistry involves sacrificing animals and removing brain tissue. If someone has a genetically modified mouse that they want us to take measurements on, it is difficult to send cells that stay intact overseas. We have a room on our campus that can be sterilized, where we can complete the dissection and run samples without having to quarantine the animals. In another recent example, we were collaborating with someone in the UK who was researching salt loading in animals and how this affects a certain brain region. His work was mostly conducted on rodents, but he also had a partnership with scientists in the Middle East completing similar studies on camels. The first set of samples had too high a measurement variability and so one of my senior scientists went to the Middle East to help them collect more rigorous and quantifiable samples. We find we often have to work with those collecting the samples and train them to obtain the best quality data.

The Takeover of the Sweedler Group by Mass Spectrometry

Early on in Professor Sweedler’s career at UIUC, mass spectrometry was just one of a range of analytical methods employed to take measurements of samples. The methods and equipment of mass spectrometry have since enjoyed substantial improvement and growth, allowing the method to overtake other areas.

When joined with other methods, including transcript measurements, electrophysiology and fluorescent microscopy, mass spectrometry becomes an even more powerful tool for researchers. Professor Sweedler outlined the ways in which analytical measurements have developed through the quarter century since he started at the university:

My initial research proposal was on how to measure cell-to-cell signaling, in the brain. I proposed working with sea slugs, a simple organism with 10,000 neurons that can learn to find food and avoid being eaten, which we still work on today. We also now work on both simpler and more complex animal models, the more complex being rodents and sometimes primates. Twenty-five years on, we’re more capable of getting information out of our samples and conducting measurements, and equipment has entirely changed. Back then I proposed to use capillary electrophoresis with fluorescent detection, in addition to radioactivity detection and NMR. Now almost all measurements involve mass spectrometry... Somewhere along the way we started working with Bruker and as mass spectrometry improved, other methods have been used much less often.

New Instruments for New Measurements

The realm of single cell biology is quickly growing in recognition and usage, and will continue to do so in the future. This increased traction can aid in developing the improvements in the technology necessary to make these measurements.

One of the Sweedler group’s recent innovations was the development of a high throughput process, in which they scatter thousands of cells on a microscope slide, make them fluorescent, and employ optical microscopy to detect cell locations.

This data on the exact placements guides the Bruker ultrafleXtreme™ and the solariX XR™ to obtain mass spectra from the cells alone, rather than including empty spaces surrounding them. This allows them to acquire lipid and peptide profiles from tens of thousands of cells.

The Sweedler group is also aiming to develop a method to combine mass spectrometry with other chemically information-rich methodologies:

We're working with a couple of groups where we can image a brain slice with vibrational spectroscopy such as infrared and Raman. Light is non-destructive, so once we get that chemical information, we can then carry out mass spectrometry imaging. We are currently working on how to combine the different datasets.

On top of this, the team is also working on a technique that will allow them to sample brain slice content following mass spectrometry imaging at chosen locations, and subsequently carry out capillary electrophoresis mass spectrometry analyses of collected analytes.

How Has Bruker’s Technology Helped to Solve These Challenges?

There is not always an obvious answer when determining which instruments are appropriate for a specific study.

“Sometimes one instrument will excel, and another will not,” states Professor Sweedler. “There are several flavors of instruments, but when it comes to mass spectrometry imaging with MALDI MS, Bruker has very strong platform performance."

In addition to this, Bruker offers a wealth of knowledge, from software update assistance to on-site training at UIUC, and, “offers a partnership which is worth a lot!” – adds Professor Sweedler. Furthermore, Bruker enables the manner in which Professor Sweedler tests the boundaries of their instruments to ever higher limits:

We often use the instruments slightly differently than most users, which can be tricky, but Bruker has always helped us.

One job of an academic analytical chemist is to push the measurement science beyond what it can easily do. A frequent question we ask ourselves is ‘we want to make this measurement, but how can we make the instrument do it?’ – working with Bruker enables us to collect such data.

A recent procurement of Professor Sweedler’s lab was Bruker’s solariX XR™:

If I had to pick one instrument to save in a fire (and did not have to worry about its size or weight), it would be the solariX XR™... It’s a lower throughput instrument than the ultrafleXtreme™, but it provides great chemical information with the added capability of electrospray.

Future

Over the last twenty years, resolutions to the challenge of single cell measurements have altered. Professor Sweedler explains that:

Companies like Bruker are enabling for single cell measurements because they are producing increasingly high quality mass spectrometers... It’s kind of a fun evolution! The last two years have been especially exciting with the combination of mass spectrometry with other approaches. In the next decade, I would like to see more robust methods of data integration with other non-mass spectrometry based platforms. We can currently acquire a large amount of data, but the ability to integrate data not just with Bruker instruments but with other instrumental platforms as well, really needs to be improved. Data integration tools need to become more universal.

The endurance of the Sweedler group’s relationship with Bruker is a great endorsement for Bruker’s high specification instrumentation, which enables labs such as Professor Sweedler’s to continue to make ground-breaking discoveries by testing the equipment to its limits.

References

1. National Science Foundation, 2016. Sea slug brain chemistry reveals a lot about human memory, learning www.nsf.gov/news/special_reports/science_nation/ sluggishthoughts.jsp [Accessed 16/01/2017]

About Bruker Daltonics

Discover new ways to apply mass spectrometry to today’s most pressing analytical challenges. Innovations such as Trapped Ion Mobility (TIMS), smartbeam and scanning lasers for MALDI-MS Imaging that deliver true pixel fidelity, and eXtreme Resolution FTMS (XR) technology capable to reveal Isotopic Fine Structure (IFS) signatures are pushing scientific exploration to new heights. Bruker's mass spectrometry solutions enable scientists to make breakthrough discoveries and gain deeper insights.


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Last updated: Apr 2, 2019 at 10:12 AM

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