Abstract
The purpose of this study was to establish a quantitative proteomic method able to accurately quantify pathological changes in the protein expression levels of not only non-membrane proteins, but also membrane proteins, using formalin-fixed paraffin-embedded (FFPE) samples. Protein extraction from FFPE sections of mouse liver was increased 3.33-fold by pressure cycling technology (PCT) and reached the same level as protein extraction from frozen sections. After PCT-assisted processing of FFPE liver samples followed by SWATH-MS-based comprehensive quantification, the peak areas of 88.4% of peptides agreed with those from matched fresh samples within a 1.5-fold range. For membrane proteins, this percentage was remarkably increased from 49.1 to 93.8% by PCT. Compared to the conventional method using urea buffer, the present method using phase-transfer surfactant (PTS) buffer at 95 °C showed better agreement of peptide peak areas between FFPE and fresh samples. When our method using PCT and PTS buffer at 95 °C was applied to a bile duct ligation (BDL) disease model, the BDL/control expression ratios for 80.0% of peptides agreed within a 1.2-fold range between FFPE and fresh samples. This heat-compatible FFPE-PCT-SWATH proteomics technology using PTS is suitable for quantitative studies of pathological molecular mechanisms and biomarker discovery utilizing widely available FFPE samples.
Highlights
The purpose of this study was to establish a quantitative proteomic method able to accurately quantify pathological changes in the protein expression levels of non-membrane proteins, and membrane proteins, using formalin-fixed paraffin-embedded (FFPE) samples
To verify the usefulness of the FFPE-pressure cycling technology (PCT)-SWATH proteomics protocol established in the present study for studies of pathological molecular mechanisms and biomarker discovery, we examined whether the pathological changes in protein expression levels quantified in FFPE sections by means of FFPE-PCT-SWATH proteomics quantitatively agree with those in fresh tissue, using bile duct ligation (BDL) mouse liver as a disease model tissue (Fig. 4 and Supplementary Tables 7–10)
When we focused on cytosol proteins, FFPE-PCT-SWATH proteomics increased the percentage (77.2%, Supplementary Fig. 7a) of tryptic peptides whose pathological changes in expression level in FFPE samples lay within a 1.2-fold range of those in fresh samples, as compared to the conventional method (65.2%, Supplementary Fig. 7d)
Summary
The purpose of this study was to establish a quantitative proteomic method able to accurately quantify pathological changes in the protein expression levels of non-membrane proteins, and membrane proteins, using formalin-fixed paraffin-embedded (FFPE) samples. After PCT-assisted processing of FFPE liver samples followed by SWATH-MS-based comprehensive quantification, the peak areas of 88.4% of peptides agreed with those from matched fresh samples within a 1.5-fold range. For membrane proteins, this percentage was remarkably increased from 49.1 to 93.8% by PCT. When our method using PCT and PTS buffer at 95 °C was applied to a bile duct ligation (BDL) disease model, the BDL/control expression ratios for 80.0% of peptides agreed within a 1.2fold range between FFPE and fresh samples This heat-compatible FFPE-PCT-SWATH proteomics technology using PTS is suitable for quantitative studies of pathological molecular mechanisms and biomarker discovery utilizing widely available FFPE samples. Sequential window acquisition of all theoretical fragment ion spectra–mass spectrometry (SWATH-MS) is the latest quantitative comprehensive proteomics method, and is highly accurate and reproducible compared to conventional shotgun proteomics[6]
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