Background
To extract proteins from formalin-fixed paraffin embedded (FFPE) tissue most protocols use high concentrations of SDS with heating for >1 hour to 100 °C. Two widely employed methods (SP3 bead capture and FASP) are able to mostly remove SDS, although we found that even minute amounts of remaining SDS alter chromatographic retention times. This is detrimental for run-run reproducibility, especially for data-independent acquisition (DIA) and could compromise quantitation from multi-batch cohort studies. Here, we developed an improved sample preparation workflow and DIA strategy aimed at supporting robust quantitative analysis of colorectal cancer (CRC) FFPE specimens.
Methodologies
CRC FFPE sections (5x5 µm per sample) were macro-dissected, lysed and proteins reduced and alkylated. Proteins were digested and purified according to a reported SP3 protocol (PMID: 29565595) with subsequent SDS precipitation using KCl. A pooled sample was used to acquire 6 small window DIA gas-phase fractions for chromatogram library generation. Wide window DIA data were acquired using an overlapping window scheme. All data were acquired on a Thermofisher QExactive HFX mass spectrometer with 140min LC time using a 50cm x 75µm self-packed pulled column.
Results
Using this method we observed highly reproducible chromatography from FFPE extracted samples. We could measure all FFPE samples reproducibly and with minimal RT shifts from the library (median deltaRT: 1.30 ±0.16 min). The gas-phase fractionation library consisted of over 6000 proteins and when applied to the EncyclopeDIA workflow (PMID: 30510204) allowed for the Identification and quantification of >4000 proteins in each of the 12 test specimens from ~1ug on column load.
Conclusion
Combining SP3 bead clean-up with a subsequent precipitation of residual SDS led to highly reproducible RT across FFPE extracted samples. Using the EncyclopeDIA workflow we were able to acquire chromatogram libraries with similar depth to that of high pH fractionation with less sample handling.