Parallel Analysis of the Proteome, Histone PTMs & RNA Modifications from Frozen & FFPE Tissues by MS
Presented by Joanna Lempiainen, Ph.D.
Postdoctoral Research Scholar, Washington University School of Medicine (Saint Louis, MO) at ASMS 2023
Acquiring reliable proteomics and epigenetics data from limited volumes of tissue is essential for understanding dysregulated signaling in diseases such as malignant peripheral nerve sheath tumor (MPNST). Here, we develop a workflow to simultaneously characterize the total proteome, histone PTMs, and RNA modifications from frozen sections and formalin-fixed, paraffin-embedded (FFPE) core punches of MPNST tissue.
FFPE core punches of 5 mg were lysed by sonication with adaptive focused acoustic (AFA) sonication (Covaris) in 5% SDS. For total proteome, proteins were digested using S-Trap (Protifi) and analyzed by MS. For histone extraction, 200 µg of the total protein was loaded in adjacent wells of acrylamide SDS-PAGE gel, histone bands excised, and in-gel propionylated and trypsinized before MS analysis. For RNA modifications, RNA was extracted from the sonicated cell lysate using TRIzol and RNA processed to single nucleosides and permethylated before MS analysis. Frozen sections of 5-300 mg were prepared with Quick-RNA Tissue Microprep kit (Zymo Research), where total proteome was acetone precipitated from the flow-through and RNA eluted from the column. Total proteome, histones and RNA were otherwise prepared as for the FFPE punches.
FFPE punches and frozen sections yielded 600-700 µg and 400-2800 µg of protein, respectively. MS analysis using Exploris 240 (Thermo Scientific) in DDA mode quantified on average 2200 proteins in each sample and on the ZenoTOF 7600 (SCIEX) in DIA mode on average 3500 proteins per sample. Proteins were identified from different cellular compartments with about 40% cytoplasmic, 30% nuclear, 10% cell membrane 10% secreted and 10% mitochondrial proteins. RNA yields after cleanup were from FFPE samples 1000-3000 ng and frozen tissues 300-700 ng. For RNA modifications, MS analysis successfully identified m6A in FFPE samples and m6A, i6A and t6A in frozen sections. In addition, MS successfully quantified more than 100 single and combinatorial histone PTMs such as methylations of H3K27 and H3K36 from the gel-excised histones.
These workflows are promising for parallel analysis of the total proteome, histone PTMs, and RNA modifications from tissue by MS.
Видео Parallel Analysis of the Proteome, Histone PTMs & RNA Modifications from Frozen & FFPE Tissues by MS канала Covaris, LLC
Postdoctoral Research Scholar, Washington University School of Medicine (Saint Louis, MO) at ASMS 2023
Acquiring reliable proteomics and epigenetics data from limited volumes of tissue is essential for understanding dysregulated signaling in diseases such as malignant peripheral nerve sheath tumor (MPNST). Here, we develop a workflow to simultaneously characterize the total proteome, histone PTMs, and RNA modifications from frozen sections and formalin-fixed, paraffin-embedded (FFPE) core punches of MPNST tissue.
FFPE core punches of 5 mg were lysed by sonication with adaptive focused acoustic (AFA) sonication (Covaris) in 5% SDS. For total proteome, proteins were digested using S-Trap (Protifi) and analyzed by MS. For histone extraction, 200 µg of the total protein was loaded in adjacent wells of acrylamide SDS-PAGE gel, histone bands excised, and in-gel propionylated and trypsinized before MS analysis. For RNA modifications, RNA was extracted from the sonicated cell lysate using TRIzol and RNA processed to single nucleosides and permethylated before MS analysis. Frozen sections of 5-300 mg were prepared with Quick-RNA Tissue Microprep kit (Zymo Research), where total proteome was acetone precipitated from the flow-through and RNA eluted from the column. Total proteome, histones and RNA were otherwise prepared as for the FFPE punches.
FFPE punches and frozen sections yielded 600-700 µg and 400-2800 µg of protein, respectively. MS analysis using Exploris 240 (Thermo Scientific) in DDA mode quantified on average 2200 proteins in each sample and on the ZenoTOF 7600 (SCIEX) in DIA mode on average 3500 proteins per sample. Proteins were identified from different cellular compartments with about 40% cytoplasmic, 30% nuclear, 10% cell membrane 10% secreted and 10% mitochondrial proteins. RNA yields after cleanup were from FFPE samples 1000-3000 ng and frozen tissues 300-700 ng. For RNA modifications, MS analysis successfully identified m6A in FFPE samples and m6A, i6A and t6A in frozen sections. In addition, MS successfully quantified more than 100 single and combinatorial histone PTMs such as methylations of H3K27 and H3K36 from the gel-excised histones.
These workflows are promising for parallel analysis of the total proteome, histone PTMs, and RNA modifications from tissue by MS.
Видео Parallel Analysis of the Proteome, Histone PTMs & RNA Modifications from Frozen & FFPE Tissues by MS канала Covaris, LLC
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27 июня 2023 г. 21:54:16
00:18:42
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