Optimization of Extraction Methods for the Quantification of Proteins in Mammalian Tissues

Abstract

With the development of new vaccine technologies, such as mRNA vaccines, tissue studies are becoming increasingly important. Knowledge of the antigen expression amounts and where the antigen accumulates in the body is essential for designing safe and effective vaccines. Mammalian tissues present challenges in the detection and accurate quantification of target proteins because of their complexity and the lack of protocols that efficiently extract proteins with minimal sample loss. Here, we describe a protocol for the detection and accurate quantification of protein targets in commercially available snap-frozen lung, liver, kidney, and spleen of European domestic ferrets (Mustela putorius furo) by isotope dilution mass spectrometry (IDMS). Housekeeping proteins were chosen that range in abundance to account for different masses of tissue slices of the same organ. Target peptides used for IDMS quantification were conserved across several of the common animal model systems, including baby hamster kidney, mouse, and ferret. Hemagglutinin, the primary antigen of an influenza vaccine, was added at various concentrations to demonstrate the recovery of low-abundance proteins from the complex tissue homogenate. By using housekeeping proteins and a preparation protocol that minimizes sample loss, this study shows that IDMS can accurately quantify proteins in mammalian tissues with unmatched sensitivity and specificity.