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

Sarah A. Michaud, Helena Petrosova, Angela M. Jackson, Jamie C. McGuire, Nicholas J. Sinclair, Milan Ganguly, Ann M. Flenniken, Lauryl M.J. Nutter, Colin McKerlie, David Schibli, Derek Smith, Christoph H. Borchers

Research output: Contribution to journalArticlepeer-review

Abstract

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.

Original languageEnglish
Pages (from-to)305-316
Number of pages12
JournalJournal of Proteome Research
Volume20
Issue number1
DOIs
Publication statusPublished - 1 Jan 2021

Keywords

  • acetone precipitation
  • homogenization
  • mass spectrometry
  • sample preparation
  • tissue lyophilization
  • tissue proteomics

Fingerprint

Dive into the research topics of 'Process and Workflow for Preparation of Disparate Mouse Tissues for Proteomic Analysis'. Together they form a unique fingerprint.

Cite this