Peptide Puzzle Pieces: Perceiving Peculiarly Produced Proteins

Jackson Eberwein, Sonoma State University

The Approach: In my previous post, I discussed some of the problems about Domoic Acid Toxicosis in California sea lions, closing with the potential benefit of finding a biomarker protein. The first step in finding anything is to take a good look! How does someone look at something as small as a protein, though? When trying to see which proteins are in a sample of blood plasma and count how many there are of each, it can get complicated. The way I am achieving this is by using an instrument called a mass spectrometer. With it, I can predict the identity and amount of each protein in a sample. The mass spectrometer is picky, however. To read my proteins, it wants them to be cut up first. 

Orbitrap mass spectrometer (right) with attached liquid chromatograph (left).

Proteins are similar in structure to a tangle of string. The tangle first has to be unraveled, then it is cut up into small bits called peptides. These peptides are put into the mass spectrometer to be read. The shape of each peptide is pretty unique, and that unique shape is used to detect and measure them. With the help of some very handy computer programs, peptide measurements can be compared to a California sea lion genetic database to predict the protein that each peptide came from and how many of those proteins there might have been in the sample. Once we have the names and amounts of the proteins in the sea lion samples, the protein differences between each sample can be looked at. This is where we look for our biomarker. If there are one or more proteins that appear at consistently different levels in sea lion samples with Domoic Acid Toxicosis than in samples without it, those proteins have potential as good biomarkers! 

Acknowledgements

I would like to thank Dr. Michael Janech, Dr. Benjamin Neely, Alison Bland, The Marine Mammal Center, & College of Charleston. Supported in part by the Fort Johnson REU Program, NSF DBI-1757899.

Advertisements

How to Train Your Shewanella

Katherine Mateos, Carleton College

The Approach: In my previous post, I introduced my project, investigating the role of Antarctic bacterium, Shewanella BF02, in the cycling of volatile organic sulfur compounds (VOSCs). 

Sterile technique in action Photo Credit: Peter Lee

The first order of business in this effort is keeping the Shewanella alive and happy. In order to do this in the lab, I make a liquid (known in the biology world as “medium”) for the Shewanella to live in. Our medium is designed to resemble Blood Falls in chemical makeup. In particular, it is very salty, and contains iron and sulfate. I am also careful to remove all the dissolved oxygen in the medium, since the Blood Falls water has very little oxygen. In my medium, I am also careful to keep out any bacteria other than my Shewanella. Since microbes are everywhere, including in the air, on my skin, and on the lab bench, I  use a special set of techniques to avoid unwanted bacteria from infecting my samples. 

Membrane Inlet Mass Spectrometer

Once we have a perfect mix of chemicals for Shewanella, I also add my target organic sulfur compounds. Because I want to see if Shewanella changes these added compounds, I keep track of them using a technique called isotope labeling. Isotope labeling is a clever trick, where the target compounds are tagged with atoms that are the tiniest bit heavier than the ones that we usually see. If Shewanella make the labeled compounds into the VOSC products that I am interested in, those products will also have the same tag, making it easy to identify them.

To identify the tiny differences in mass between tagged and untagged molecules, I use a piece of equipment called a mass spectrometer. A mass spectrometer works kind of like a scale and can determine the mass of each molecule. This allows me to detect isotopically labeled VOSC products. If I see isotopically labeled products, I can be pretty sure that the Shewanella are cycling the labeled compound that I added to their medium. 

Thank you to my mentor, Dr. Peter A. Lee, and our collaborators, Dr. Jill Mikucki and Abigail Jarratt, for their guidance in the research process. This project is supported by the Fort Johnson REU Program, NSF DBI-1757899.

Brrrrr…..

Kaylie Anne Costa, University of Miami

The problem: Do you hate the cold? Well manatees can’t stand it either! Every year Florida manatees (e.g., West Indian Manatee, Trichechus manatus latirostris) migrate to warmer waters during the winter months. In the past, they have used locations such as springs, layers of warm water created by salinity anomalies, and even the effluents from coastal power plants to stay warm. Unfortunately, new developments and recreational activities are taking over the natural warm water sources and most of the power plants are shutting down, so manatees have no haven for warm water.

ctaylor_wildlife_trust_1-26-2005-2_500

Manatees utilize the warm effluent water of a coastal power plant in Riviera Beach, Florida (Source)

When water temperatures fall below 20 °C (68°F), Florida manatees become susceptible to cold stress syndrome (CSS), which is a breakdown of normal biological and immunological processes that often leads to death (Bossart 2001). Manatees experiencing CSS have characteristic lesions and other symptoms like emaciation, lethargy, fat atrophyand loss, epidermal hyperplasia, pneumonia, and myocardial degeneration (Bossart, 2001; Bossart et al., 2003). CSS plays a major role in major manatee die off events and the number of cases continues to increase as manatees lose more and more warm water refuges to development and recreation. I will be expanding the current scientific knowledge of CSS by analyzing the lipids (aka fats) and metabolites, which are the products remaining after biological processes such as digestion, respiration, maintaining homeostasis, etc. in manatee plasma samples using mass spectrometry in hopes of learning more about metabolism for therapeutic applications.

Protecting Florida manatees is important for so many reasons. First off, the US Endangered Species act listed the Florida manatee as endangered in 2001, but recently reduced their status to only threatened in 2017 (Public Affairs Office, 2018). Without intervention, this species could easily return to its endangered status. Secondly, marine mammals are great sentinels to model how environmental changes will impact human health due to their physiological similarities, long life spans, and thick blubber’s ability to store large amounts of contaminants (Bossart, 2011). Thirdly, manatees help control the growth of sea grass beds. The presence of healthy sea grass beds allows the ecosystems around them to thrive. Lastly, manatees support economies through ecotourism. This research is necessary to protect Florida manatees from this understudied condition.

A huge thank you to my mentor Dr. John Bowden and co-mentor Dr. Mike Napolitano as well as everyone at NIST for all of their help and guidance. I would also like to thank the National Science Foundation for funding and the Fort Johnson REU program for making this research possible (NSF DBI-1757899).

References:

Public Affairs Office. (2018, February 7). Florida Manatee -Issues and Information. Retrieved June 17, 2018, from https://www.fws.gov/northflorida/manatee/manatees.htm

Bossart, G. (2001) Manatees. In: L. Dierauf & F. Gulland (eds.) Marine Mammal Medicine, pp. 939–960. CRC Press, Boca Raton, FL.

Bossart, G. D., Meisner, R. A., Rommel, S. A., Ghim, S. J., & Jenson, A. B. (2003). Pathological features of the Florida manatee cold stress syndrome. Aquatic Mammals, 29(1), 9–17.

Bossart, G. D. (2011). Marine mammals as sentinel species for oceans and human health. Veterinary Pathology, 48(3), 676–690.

Manatees and PFCs- The Future of Contaminant Studies

Kady Palmer, Eckerd College

IMG_1115

In my previous post, “The Problem with PFCs- Seeking Answers in Plasma”, the abundance of perfluorinated chemicals, or more specifically perfluoroalkyl acids (PFAAs), was analyzed in manatee plasma and correlated to variables such as site, water temperature, and blood chemistry. The purpose of this study was to develop a greater understanding of these chemical contaminants in regards to their routes of exposure and subsequent health effects.

Accumulation of PFAAs within organisms is proposed to be predominantly attributed through diet. Therefore, apex predators, like alligators, dolphins, and humans are found to be at a higher risk for increased concentrations of these chemicals in their body (Bangma et al., 2017, Fair et al., 2012). This is a result of biomagnification, or increasing levels of a compound as one continues up the food chain or trophic hierarchy. Manatees, however, are not predators, and are considered lower on the trophic hierarchy due to their herbivorous diet. With that knowledge, the amount of PFAAs within them, if any, was hypothesized to be very small.

After obtaining data from chemical extractions and liquid chromatography tandem mass spectrometry (LC-MS/MS), concentrations of at least two perfluoroalkyl acids were detected in all 69 manatee plasma samples. What that means is that PFAAs are integrating into the biological systems of manatees and accumulating within their bloodstream, presenting different results than our initial hypothesis.

pfos

One of the most common PFAAs found in manatee plasma, known as perfluorooctanesulfonic acid (PFOS). Photo from: http://pubs.sciepub.com/ces/2/1/3/

Data and statistical analyses determined location-based differences in PFAA concentrations. In addition, correlations were found between high PFAA burden, blood chemistry measurements, and water temperature at the time of sampling. With this information, a basis for further investigations is possible to begin determining potential health effects of PFAAs in not only manatees, but in humans as well.

manateesCR

Because manatees cannot tolerate cold water, they congregate in warm waters during the winter seasons. Interestingly, correlations between water temperatures and PFAA values were found in this study. Photo from: http://proof.nationalgeographic.com/2014/07/21/floridas-manatees-the-search-for-warmer-water/

In summary, the purpose of this experiment was to answer two questions: 1) Are PFAAs present in manatee plasma? 2) If so, can heavy burdens of PFAAs be statistically correlated to health variables?

The first question was answered within the first week of analysis, simply by identifying detectable levels of these chemicals in manatee plasma. The second question, however, is more complicated to answer. The statistics say that there are associations between PFAAs and differing health measurements, however, the significance and meaning of that data is something future research must focus on. The reasons behind the correlations are still unknown, even though some explanations may be proposed.

I would like to extend an enormous thank you to everyone who made this project possible, including Dr. Jacqueline Bangma, Dr. Jessica Reiner, and my extremely motivating mentor, Dr. John Bowden. I would also like to thank the National Science Foundation for their funding, the College of Charleston’s Grice Marine Lab for hosting this REU, and the USGS Sirenia project for supplying the samples I utilized in this project.

References:

Bangma, Jacqueline T., John A. Bowden, Arnold M. Brunell, Ian Christie, Brendan Finnell, Matthew P. Guillette, Martin Jones, et al. “Perfluorinated Alkyl Acids in Plasma of American Alligators (Alligator Mississippiensis) from Florida and South Carolina.” Environmental Toxicology and Chemistry, no. 4 (2017a): 917. doi:10.1002/etc.3600.

Fair, Patricia A., Magali Houde, Thomas C. Hulsey, Gregory D. Bossart, Jeff Adams, Len Balthis, and Derek C.G. Muir. “Assessment of Perfluorinated Compounds (PFCs) in Plasma of Bottlenose Dolphins from Two Southeast US Estuarine Areas: Relationship with Age, Sex and Geographic Locations.” Marine Pollution Bulletin 64 (January 1, 2012): 66–74. doi:10.1016/j.marpolbul.2011.10.022.

 

The Problem with PFCs- Seeking Answers in Plasma

Kady Palmer, Eckerd College

Manatee_CR

I previously outlined the problem of perfluorinated chemicals (PFCs) in the environment and their unknown health effects.  In order to gain this knowledge, it is essential to determine what types of PFCs are frequently used and the mechanisms by which an individual would be exposed to them. Here, we are measuring the presence or absence of 15 PFCs that are commonly associated with non-stick cookware, firefighting foam, and water-resistant materials.

This compiled list of PFCs is the basis of my research procedure. From here, I must learn how these compounds interact with biological components in organisms in order to understand their subsequent health effects. With that being said, the type of samples I am analyzing is a topic worth explaining. PFCs are known to be “proteinophilic” or, attracted to proteins in the bloodstream of organisms like humans and, in the case of my study, manatees. Therefore, I am using manatee plasma to test for the total individual burden of PFCs. 

PFAAs1       PFAAS2

Fig 1. 69 collection tubes containing manatee plasma samples (left). Aliquots of 22 samples of manatee plasma for future studies (right). Photos taken by me!

With 69 different plasma samples, I am performing a series of procedures that allow me to extract the PFCs. After completing multiple chemical processes (methodology proposed by Reiner et al., 2012), I am left with a liquid (containing the PFCs), measuring no more than 1 mL to be placed into a small vial. From here the vials are inserted into a liquid chromatography tandem mass spectrometer (LC-MS/MS), a machine that reads each of the 15 unique chemical structures of the outlined PFCs of interest and determines their abundance in each vial. This system isolates the concentration of each perfluorinated chemical for every one of the 69 manatee samples.

Mass Spec

Fig 2. The basic process a mass spectrometer performs in order to provide the concentration of chemicals being studied. Photo from: http://www.emdmillipore.com/US/en/water-purification/learning-centers/Anwendungen/organic-analysis/lc-ms/lWib.qB.vb4AAAFA5fIBvVBh,nav?ReferrerURL=https%3A%2F%2Fwww.google.com%2F&bd=1

The concentrations of these chemicals is the ultimate goal of my research study. This data will be compared to manatee location, morphometrics, body condition, sex, and more, in order to gain a better understanding of the overall PFC burden on these animals. These factors, or variables, may also provide insight into what may be influencing the burden intensity an individual may face. Once this knowledge is gathered, potential links to the health effects of PFC accumulation can be investigated in both manatees and humans.

I’d like to thank the National Science Foundation for funding this research opportunity and the College of Charleston’s Grice Marine Laboratory REU program for making this experience possible. A special thanks to the NIST team who has been teaching and supporting me throughout this process, specifically, Dr. Jessica Reiner, Jacqueline Bangma, and my mentor, Dr. John Bowden.

Note: These samples were collected as part of a health assessment of manatees by the USGS Sirenia Project. No manatees were harmed in the process of obtaining them.

References

Reiner, Jessica, Karen Phinney, and Jennifer Keller. “Determination of Perfluorinated Compounds in Human Plasma and Serum Standard Reference Materials Using Independent Analytical Methods.” Analytical & Bioanalytical Chemistry 401, no. 9 (January 15, 2012): 2899–2907. doi:10.1007/s00216-011-5380-x.z