Protein Turnover Dynamics Analysis With Subcellular Spatial Resolution
Protein synthesis and degradation (i.e., turnover) forms an important part of protein homeostasis and has been implicated in many age-associated diseases. Different cellular locations, such as organelles and membraneless compartments, often contain individual protein quality control and degradation...
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| Format: | Article |
| Language: | English |
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Bio-protocol LLC
2025-08-01
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| Series: | Bio-Protocol |
| Online Access: | https://bio-protocol.org/en/bpdetail?id=5409&type=0 |
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| author | Lorena Alamillo Alexander Black Maggie Lam Edward Lau |
| author_facet | Lorena Alamillo Alexander Black Maggie Lam Edward Lau |
| author_sort | Lorena Alamillo |
| collection | DOAJ |
| description | Protein synthesis and degradation (i.e., turnover) forms an important part of protein homeostasis and has been implicated in many age-associated diseases. Different cellular locations, such as organelles and membraneless compartments, often contain individual protein quality control and degradation machineries. Conventional methods to assess protein turnover across subcellular compartments require targeted genetic manipulation or isolation of specific organelles. Here we describe a protocol for simultaneous proteome localization and turnover (SPLAT) analysis, which combines protein turnover measurements with unbiased subcellular spatial proteomics to measure compartment-specific protein turnover rates on a proteome-wide scale. This protocol utilizes dynamic stable isotope labeling of amino acids in cell culture (dynamic SILAC) to resolve the temporal information of protein turnover and multi-step differential ultracentrifugation to assign proteins to multiple subcellular localizations. We further incorporate 2D liquid chromatography fractionation to greatly increase analytical depth while multiplexing with tandem mass tags (TMT) to reduce acquisition time 10-fold. This protocol resolves the spatial and temporal distributions of proteins and can also reveal temporally distinct spatial localizations within a protein pool. |
| format | Article |
| id | doaj-art-74a9129eba9e411ea8aaec0cd6ce327f |
| institution | DOAJ |
| issn | 2331-8325 |
| language | English |
| publishDate | 2025-08-01 |
| publisher | Bio-protocol LLC |
| record_format | Article |
| series | Bio-Protocol |
| spelling | doaj-art-74a9129eba9e411ea8aaec0cd6ce327f2025-08-20T03:02:52ZengBio-protocol LLCBio-Protocol2331-83252025-08-01151510.21769/BioProtoc.5409Protein Turnover Dynamics Analysis With Subcellular Spatial ResolutionLorena Alamillo0Alexander Black1Maggie Lam2Edward Lau3Deptartment of Medicine, University of Colorado School of Medicine, Aurora, CO, USADeptartment of Medicine, University of Colorado School of Medicine, Aurora, CO, USADeptartment of Medicine, University of Colorado School of Medicine, Aurora, CO, USADeptartment of Biochemistry & Molecular Genetics, University of Colorado School of Medicine, Aurora, CO, USA, Consortium for Fibrosis Research & Translation, University of Colorado School of Medicine, Aurora, CO, USADeptartment of Medicine, University of Colorado School of Medicine, Aurora, CO, USAConsortium for Fibrosis Research & Translation, University of Colorado School of Medicine, Aurora, CO, USAProtein synthesis and degradation (i.e., turnover) forms an important part of protein homeostasis and has been implicated in many age-associated diseases. Different cellular locations, such as organelles and membraneless compartments, often contain individual protein quality control and degradation machineries. Conventional methods to assess protein turnover across subcellular compartments require targeted genetic manipulation or isolation of specific organelles. Here we describe a protocol for simultaneous proteome localization and turnover (SPLAT) analysis, which combines protein turnover measurements with unbiased subcellular spatial proteomics to measure compartment-specific protein turnover rates on a proteome-wide scale. This protocol utilizes dynamic stable isotope labeling of amino acids in cell culture (dynamic SILAC) to resolve the temporal information of protein turnover and multi-step differential ultracentrifugation to assign proteins to multiple subcellular localizations. We further incorporate 2D liquid chromatography fractionation to greatly increase analytical depth while multiplexing with tandem mass tags (TMT) to reduce acquisition time 10-fold. This protocol resolves the spatial and temporal distributions of proteins and can also reveal temporally distinct spatial localizations within a protein pool.https://bio-protocol.org/en/bpdetail?id=5409&type=0 |
| spellingShingle | Lorena Alamillo Alexander Black Maggie Lam Edward Lau Protein Turnover Dynamics Analysis With Subcellular Spatial Resolution Bio-Protocol |
| title | Protein Turnover Dynamics Analysis With Subcellular Spatial Resolution |
| title_full | Protein Turnover Dynamics Analysis With Subcellular Spatial Resolution |
| title_fullStr | Protein Turnover Dynamics Analysis With Subcellular Spatial Resolution |
| title_full_unstemmed | Protein Turnover Dynamics Analysis With Subcellular Spatial Resolution |
| title_short | Protein Turnover Dynamics Analysis With Subcellular Spatial Resolution |
| title_sort | protein turnover dynamics analysis with subcellular spatial resolution |
| url | https://bio-protocol.org/en/bpdetail?id=5409&type=0 |
| work_keys_str_mv | AT lorenaalamillo proteinturnoverdynamicsanalysiswithsubcellularspatialresolution AT alexanderblack proteinturnoverdynamicsanalysiswithsubcellularspatialresolution AT maggielam proteinturnoverdynamicsanalysiswithsubcellularspatialresolution AT edwardlau proteinturnoverdynamicsanalysiswithsubcellularspatialresolution |