Methods for precipitating plasma proteins for stable isotope analysis of elasmobranch blood

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.

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.