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Method Article
Alex Kentsis
1 Molecular Pharmacology Program, Sloan Kettering Institute, Tri-Institutional Ph.D. Program in Chemical Biology, Memorial Sloan Kettering Cancer Center, New York, New York 10065, United States. 2 Department of Pediatrics, Pharmacology, and Physiology & Biophysics, Weill Cornell Medical College, Cornell University, New York, New York 10065, United States
Corresponding Author
ORCiD: https://orcid.org/0000-0002-8063-9191
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Human and animal tissues are frequently fresh frozen for archival storage. These specimens can be used for proteomic analysis to analyze proteome composition. A critical step in these proteomic analyses is the extraction of proteins from fresh frozen samples, which can affect the sampling of the proteomes. Here, we compare three different methods for protein extraction from fresh frozen mouse heart tissue: 1) extraction using SDS buffer followed by FASP purification, 2) extraction using SDS buffer followed by S-Trap purification, and 3) extraction using a guanidine hydrochloride buffer followed by in-solution digestion. Based on bicinchoninic acid assay, all three methods display similar recovery of total protein content. However, proteomics-based analysis identified far fewer proteins from the extraction guanidine hydrochloride. SDS-FASP identifies as many proteins as the SDS-STrap method with good coverage of proteins from different cellular compartments.
Keywords
Proteomics, Tissue, Mass Spectrometry, Protein Extraction, Fresh Frozen
Figure 1
This is a list of supplementary files associated with this preprint. Click to download.
- ThreemethodFFproteomicsTable1.tif
Table 1: Comparison of protein recovery based for three different methods: 1) SDS-FASP, 2) SDS-Strap, 3) GH-IS.
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An open repository of community-contributed protocols sponsored by Nature Research.
Learn more about Protocol Exchange
Method Article
Alex Kentsis
1 Molecular Pharmacology Program, Sloan Kettering Institute, Tri-Institutional Ph.D. Program in Chemical Biology, Memorial Sloan Kettering Cancer Center, New York, New York 10065, United States. 2 Department of Pediatrics, Pharmacology, and Physiology & Biophysics, Weill Cornell Medical College, Cornell University, New York, New York 10065, United States
Corresponding Author
ORCiD: https://orcid.org/0000-0002-8063-9191
Version History
CURRENT STATUS: Posted
Version 1
Posted 22 Jul, 2020
No community comments so far
Metrics
Comments: 0
HTML Views: ...
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Human and animal tissues are frequently fresh frozen for archival storage. These specimens can be used for proteomic analysis to analyze proteome composition. A critical step in these proteomic analyses is the extraction of proteins from fresh frozen samples, which can affect the sampling of the proteomes. Here, we compare three different methods for protein extraction from fresh frozen mouse heart tissue: 1) extraction using SDS buffer followed by FASP purification, 2) extraction using SDS buffer followed by S-Trap purification, and 3) extraction using a guanidine hydrochloride buffer followed by in-solution digestion. Based on bicinchoninic acid assay, all three methods display similar recovery of total protein content. However, proteomics-based analysis identified far fewer proteins from the extraction guanidine hydrochloride. SDS-FASP identifies as many proteins as the SDS-STrap method with good coverage of proteins from different cellular compartments.
Figure 1
This is a list of supplementary files associated with this preprint. Click to download.
- ThreemethodFFproteomicsTable1.tif
Table 1: Comparison of protein recovery based for three different methods: 1) SDS-FASP, 2) SDS-Strap, 3) GH-IS.
Loading...