Journal Article Epublish
PubMed: 39667914 DOI: 10.26508/lsa.202403130
FrozONE: quick cell nucleus enrichment for comprehensive proteomics analysis of frozen tissues.
Life Sci Alliance 8(3)
Authors: Lukas A Huschet, Fabian P Kliem, Peter Wienand, Claudia M Wunderlich, Andrea Ribeiro, Isabel Bustos-Martínez, Ángel Barco, F Thomas Wunderlich, Maciej Lech, Maria S Robles
Affiliations
- Institute of Medical Psychology and Biomedical Center (BMC), Faculty of Medicine, LMU, Munich, Germany.
- Max Planck Institute for Metabolism Research, Center for Molecular Medicine Cologne (CMMC) and Policlinic for Endocrinology, Diabetes, and Preventive Medicine (PEDP), University Hospital Cologne, Cologne, Germany.
- LMU Klinikum, Medizinische Klinik und Poliklinik IV, Ludwig-Maximilians-Universität München, Munich, Germany.
- Instituto de Neurociencias (Universidad Miguel Hernández-Consejo Superior de Investigaciones Científicas), Alicante, Spain.
- Institute of Medical Psychology and Biomedical Center (BMC), Faculty of Medicine, LMU, Munich, Germany.
Abstract
Subcellular fractionation allows for the investigation of compartmentalized processes in individual cellular organelles. Nuclear enrichment methods commonly employ the use of density gradients combined with ultracentrifugation for freshly isolated tissues. Although it is broadly used in combination with proteomics, this approach poses several challenges when it comes to scalability and applicability for frozen material. To overcome these limitations, we developed FrozONE (Frozen Organ Nucleus Enrichment), a nucleus enrichment and proteomics workflow for frozen tissues. By extensively benchmarking our workflow against alternative methods, we showed that FrozONE is a faster, simpler, and more scalable alternative to conventional ultracentrifugation methods. FrozONE allowed for the study, profiling, and classification of nuclear proteomes in different tissues with complex cellular heterogeneity, ensuring optimal nucleus enrichment from different cell types and quantitative resolution for low abundant proteins. In addition to its performance in healthy mouse tissues, FrozONE proved to be very efficient for the characterization of liver nuclear proteome alterations in a pathological condition, diet-induced nonalcoholic steatohepatitis.
MeSH
Cells, Biological Science Disciplines, Non-Alcoholic Fatty Liver Disease, Workflow, Proteomics, Proteome, Tissues, Cell Nucleus