Methods for precipitating plasma proteins for stable isotope analysis of elasmobranch blood
Data files
Mar 12, 2024 version files 8.83 KB
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Isotope_Data_Sheet_w_refs.csv
4.82 KB
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README.md
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Abstract
Stable isotope analysis is a useful tool for studying the ecology of elasmobranchs. Analysis of elasmobranch blood plasma provides insight into an individual’s ecology on a small temporal scale. However, plasma is a systemic transport vessel containing many dissolved constituents in variable amounts, which may bias analyses and ecological conclusions based on that data.
In this study, we develop a new method of protein precipitation using ethanol and acetonitrile to isolate the protein fraction of plasma from Sandbar Sharks, and examine its effects on carbon and nitrogen stable isotope values. We also tested these solvent precipitations on bovine serum albumin as a control to assess the introduction of exogenous sources of C and N.
Protein isolation resulted in a significant decrease in δ13C values and a significant increase in C:N compared to untreated plasma. Isolated proteins were not significantly different in δ15N value compared to untreated plasma. We observed no change in isotope composition in bovine serum albumin samples, indicating protein precipitation does not itself affect isotope analysis.
These results suggest that the preparation of blood plasma is necessary for stable isotope analysis, to eliminate the biasing effects of other dissolved compounds. We find that solvent precipitation is an effective method of isolating proteins for stable isotope studies.
README
GENERAL INFORMATION
1. Title of Dataset: Using protein isolation on elasmobranch blood plasma for ecological research and stable isotope analysis
2. Link to Dataset: https://doi.org/10.5061/dryad.cjsxksndd
3. Author Information
a. Principal Investigator Contact Information
Name: Alexander Rubin
Institution: IBSS Corporation in Support of NOAA/NMFS
Address: Narragansett, RI USA
Email: alex1rubin@gmail.com
b. Co-investigators Contact Information
Name: Sunita Shah Walter
Institution: University of Delaware
Address: Lewes, DE USA
Email: suni@udel.edu
Name: Aaron Carlisle
Institution: University of Delaware
Address: Lewes, DE USA
Email: carlisle@udel.edu
Name: John Mohan
Institution: University of New England
Address: Biddeford, ME USA
Email: jmohan@une.edu
4. Date of data collection: 2021 – 2023
5. Geographic location of data collection: Delaware Bay, DE and University of Delaware, Newark, DE
6. Sources of funding supporting collection of the data: University of Delaware Undergraduate Research Program
SHARING/ACCESS INFORMATION
1. Licenses/restrictions placed on the data: None
2. Links to publications that cite or use the data:
Rubin, A. M., Shah Walter, S. R., Carlisle, A. B., Mohan, J. A. (2024). Using protein isolation on elasmobranch blood plasma for ecological research and stable isotope analysis. Ecology and Evolution
3. Links to other publicly accessible locations of the data: None
4. Links/relationships to ancillary data sets: None
5. Was data derived from another source? No
6. Recommended citation for this data set:
Rubin, A. M., Shah Walter, S. R., Carlisle, A. B., Mohan, J. A. (2024). Using protein isolation on elasmobranch blood plasma for ecological research and stable isotope analysis. Dryad Digital Repository. https://doi.org/10.5061/dryad.cjsxksndd
DATA & FILE OVERVIEW
1. File List:
A) Isotope Data Sheet w refs.csv
2. Relationship between files, if important: None
3. Additional related data collected that was not included in the current data package: None
4. Are there multiple versions of the dataset? No
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DATA-SPECIFIC INFORMATION FOR: Isotope Data Sheet w refs.csv
1. Number of Variables: 7
2. Number of cases/rows: 80
3. Variable List:
· SampleID: ID of an individual shark or protein sample
· Treatment type: The experimental group of a sample (ethanol treated, acetonitrile treated, untreated bulk, or reference)
· Delta 13C (ppt): Isotope ratio of carbon-13 to carbon-12 reported relative to the VPDB standard, reported in parts-per-thousand
· Mass Fraction C (%): The percent mass of carbon in the sample
· Delta 15N (ppt): Isotope ratio of nitrogen-15 to nitrogen-14 reported relative to air, reported in parts-per-thousand
· Mass Fraction N (%): The percent mass of nitrogen in the sample
· Amount (ug): Total mass of the sample analyzed, reported in micrograms
4. Missing data codes: n/a is used when measurements were not taken for a specific variable
5. Specialized formats or other abbreviations used: None
CODE/SOFTWARE
All data analysis were performed in Microsoft Excel and in R (version 4.2.2, 2022). Data were organized and mean comparisons were performed using the package dplyr (version 1.1.2, 2023).
Methods
This dataset was collected from Sandbar shark blood plasma and bovine serum albumin (BSA). Dissolved proteins were isolated from the supernatant with two solvent precipitations to be compared. Plasma and BSA samples were subject to three treatment groups: acetonitrile, ethanol, and bulk (untreated). Samples were then agitated on a vortex mixer, and centrifuged so that the protein formed a pellet in the bottom of the tube. The supernatant was removed, and the protein pellet was rinsed three times with MilliQ water to remove any remaining solvent. The protein pellet was freeze-dried, and then weighed into tin capsules for stable isotope analysis.
Isotope data was collected through stable isotope analysis using a Thermo Fisher Delta V IRMS coupled to a Costech elemental analyzer via Conflo IV interface at the University of Delaware Environmental Isotope Science Laboratory. δ13C values in this dataset are reported relative to the VPDB standard and δ15N values to air.
We used a Shapiro-Wilk test to test the normality of the isotopic data for each of our treatment groups. For normally distributed data we compared means using Analysis of Variance and Tukey HSD tests, and for non-normally distributed data we used Kruskal-Wallis and Wilcoxon signed rank tests.