Isolation of Detergent Insoluble Proteins from Mouse Brain Tissue for Quantitative Analysis Using Data Independent
Cristen Molzahn1, Lorenz Nierves2,3, Philipp F Lange2,3,4
1Michael Smith Laboratories, Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, BC, Canada.
Abstract:
Enrichment of detergent insoluble proteins is a commonly used technique for analyzing proteins that may be aggregating in disease or with age. However, various methods for enriching for these proteins are used. Here we present a method using a mild detergent (Triton X-100) and high centrifugation speed (20,000 × g) allowing for sufficient protein extraction and enrichment for large protein assemblies. Digestion is performed on columns allowing for a methanol chloroform wash to remove the highly prevalent lipids in brain tissue. This is followed by analysis by data independent acquisition mass spectrometry, which we have found to be highly reproducible. Our method is intended to enrich for amorphous aggregates, which may accumulate upon the collapse of protein homeostasis.
