Process and Workflow for Preparation of Disparate Mouse Tissues for Proteomic Analysis

We
investigated the effect of homogenization strategy and protein
precipitation on downstream protein quantitation using multiple reaction
monitoring mass spectrometry (MRM-MS). Our objective was to develop
a workflow capable of processing disparate tissue types with high
throughput, minimal variability, and maximum purity. Similar abundances
of endogenous proteins were measured in nine different mouse tissues
regardless of the homogenization method used; however, protein precipitation
had strong positive effects on several targets. The best throughput
was achieved by lyophilizing tissues to dryness, followed by homogenization via bead-beating without sample buffer. Finally, the effect
of tissue perfusion prior to dissection and collection was explored
in 20 mouse tissues. MRM-MS showed decreased abundances of blood-related
proteins in perfused tissues; however, complete removal was not achieved.
Concentrations of nonblood proteins were largely unchanged, although
significantly higher variances were observed for proteins from the
perfused lung, indicating that perfusion may not be suitable for this
organ. We present a simple yet effective tissue processing workflow
consisting of harvest of fresh nonperfused tissue, novel lyophilization
and homogenization by bead-beating, and protein precipitation. This
workflow can be applied to a range of mouse tissues with the advantages
of simplicity, minimal manual manipulation of samples, use of commonly
available equipment, and high sample quality.