I had an issue with the results concerning a bottom-up experiment using tissues preserved in FFPE.
The FFPE samples (1 kidney and 4 thymus tissues) were de-paraffinized by placing them in Covaris' microTUBE-130 AFA Fiber Screw-Caps, filled with Covaris' truXTRAC Proteins - Tissue Lysis Buffer, and processed with ultrasonication. The samples were then transferred to new tubes, heated, and centrifuged to generate a waxy paraffin layer on top. A BCA assay confirmed a presence of a decent concentration of proteins for each sample.
The next day, I proceeded to take 10 µg of two samples (1 kidney and 1 thymus) and followed the procedure outlined in Single-pot, solid-phase-enhanced sample preparation (SP3) for proteomics experiments:Article Single-pot, solid-phase-enhanced sample preparation for prot...
Reviewing the results, there were no peptide matches at all for thymus. The kidney samples were the positive control, and I am still waiting on those results. However, it is still puzzling that I got no matches. I have used the SP3 protocol previously and am confident in my handling of that workflow. So I am thinking that the issue lay somewhere with the FFPE sample preparation. My best guess is that the problem lay when I first extracted the 10 µg. De-paraffinization and SP3 were done on different days so, when I pulled the samples from the refrigerator, the thymus sample looked like the wax had resettled onto the bottom of the tube. There was still a layer of wax on the top, but there was also what looked like a not-insignificant amount on the bottom. Not remembering if this is how it had looked the previous day but definitely looking like a waxy layer, I vortexed both it and the kidney sample and centrifuged them again. Centrifugation reformed the waxy layer on top of both, but the white substance on the bottom of the thymus remained. I decided to proceed with the SP3 protocol.
Was my mistake that I vortexed the samples? Even though I could still regenerate a wax layer on top, would vortexing them mix the wax back into the samples, preventing them from appearing in the final results? I am still waiting on the kidney results, but the thymus results do not bode well. I have other thymus samples that I have not used, so I could potentially redo the SP3.
If you have not done it, the first thing you need to do is work with known samples. We never test a new technique on valuable samples. Once you are familiar and comfortable with the new technique, and know you can reliably get data, then go on to process the precious samples.
It seems you are comfortable with the SP3 protocol, so it makes sense the problem was earlier on in the process. All I can suggest is what you probably already know, go back and try again with "test" samples.
I have not used the Covaris reagents, but we routinely process FFPE samples and deparaffinisation is a standard protocol that works well in our hands. The only thing to watch out for is that all the wax is removed. Some tissue sections have a huge amount of wax and if it's not all removed it does cause problems further down the line.
Good luck
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