The main solid samples available for proteomics of cancer biopsy tissues include: Fresh-frozen (FF), OCT polymer embedded FF (FF-OCT), or formalin-fixed paraffin embedded (FFPE). Sample preparation prior to mass spectrometry (MS) requires different approaches for each. First is the time-consuming washing steps to remove OCT or paraffin. Next the tissues are lysed and proteolytically digested, and finally impurity removal by solid phase extraction (SPE). Different SPE approaches may be required for FF-OCT or FFPE since standard SPE on C18 resin does not remove polymers. In contrast, tissue lysis and digestion can be performed in common in a Barocycler.
We aimed to produce a near-universal sample preparation workflow across tissue sources. Jennifer Koh (see HUPO 2019 poster) developed an SPE method on mixed cation exchange (MCX) resin that removes OCT, allowing elimination of FF-OCT sample washing. Our first aim was to reduce the washing steps for FFPE samples. We developed a 10 min procedure for de-waxing in heated heptane then methanol that is sufficient to remove the wax, reducing washing steps by hours. Reversal of cross-linking was performed during a standard Barocycler digestion. This effectively eliminates sample washing for all typical cancer solid biopsies.
We next determined the optimum SPE method. C18 cannot be used with OCT, but both MCX and silica resins are alternatives for peptide clean-up. With all three sample sources and five rat organs representing a broad tissue density range, we found that all three SPE methods performed well. Both MCX and silica removed OCT, while all three cleaned up FF and FFPE samples. Each method was tested for reproducibility in multiple replicates.
The results show near-universal sample washing and peptide clean-up procedures, that are robust and suitable for any solid tissue source regardless of tissue fixation method.