Isolation of Detergent Insoluble Proteins from Mouse Brain Tissue for Quantitative Analysis Using Data Independent Acquisition (DIA).
| Autor: | Molzahn C; Michael Smith Laboratories, Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, BC, Canada., Nierves L; Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, BC, Canada.; Michael Cuccione Childhood Cancer Research Program, BC Children's Hospital, Vancouver, BC, Canada., Lange PF; Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, BC, Canada.; Michael Cuccione Childhood Cancer Research Program, BC Children's Hospital, Vancouver, BC, Canada.; Department of Molecular Oncology, BC Cancer, Vancouver, BC, Canada., Mayor T; Michael Smith Laboratories, Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, BC, Canada. mayor@mail.ubc.ca. |
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| Jazyk: | angličtina |
| Zdroj: | Methods in molecular biology (Clifton, N.J.) [Methods Mol Biol] 2022; Vol. 2456, pp. 29-51. |
| DOI: | 10.1007/978-1-0716-2124-0_3 |
| Abstrakt: | 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. (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.) |
| Databáze: | MEDLINE |
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