DOI: 10.5061/dryad.cjsxksndd
title: Methods for precipitating plasma proteins for stable isotope analysis of elasmobranch blood

description: 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.
description: 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. δ<sup>13</sup>C
 values in this dataset are reported relative to the VPDB standard and
 δ<sup>15</sup>N 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.
description: #### 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](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](mailto:alex1rubin@gmail.com) b.      Co-investigators Contact Information Name: Sunita Shah Walter Institution: University of Delaware Address: Lewes, DE USA Email: [suni@udel.edu](mailto:suni@udel.edu) Name: Aaron Carlisle             Institution: University of Delaware             Address: Lewes, DE USA             Email: [carlisle@udel.edu](mailto:carlisle@udel.edu) Name: John Mohan             Institution: University of New England             Address: Biddeford, ME USA             Email: [jmohan@une.edu](mailto: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](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 \############################################################################## #### 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).