Three protein extraction methods for mass spectrometry proteomics of fresh frozen mouse tissue
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.
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Table 1: Comparison of protein recovery based for three different methods: 1) SDS-FASP, 2) SDS-Strap, 3) GH-IS.
Posted 22 Jul, 2020
Three protein extraction methods for mass spectrometry proteomics of fresh frozen mouse tissue
Posted 22 Jul, 2020
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
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