I have mass spectrometry data (LC-MS/MS) from rat cortices under either drug or control treatments. The results were performed in triplicate (three pairs of rats, drug or control per pair). In addition to some of the bioinformatics analysis I am doing, I will have to validate some of the fold-changes from the mass spectrometry by Western blot in order to have my results published.
Some of the proteins I have chosen to to validate work very nicely by Western blot which captures the same trend as the mass spectrometry. However, other proteins that I have attempted to validate go in the exact opposite direction.
- In general, how accurate is mass spectrometry compared to Western blotting?
- Is it unusual to have a lot of variability between the results of the two methods? I am worried that I might have to try multiple proteins before I see the results replicated and this seems problematic to me. That leads me to the third question.
- Which result should I trust?
Other information - Some of the proteins I have chosen for validation come from lower in the list where not as many peptide fragments were detected. In some cases, fragments were detected in only 2 of the 3 biological replicates. I did not chose to validate proteins where the 2 or 3 replicates varied widely in normalized spectral count values so I would expect these readings to be accurate. Additionally, the mass spectrometry part of the experiment was performed by a reputable lab that has filtered erroneous data using FDR cutoffs. There is a high correlation overall between all three replicates for a particular treatment and low standard error between samples across the population so, statistically, I trust the mass spec results.
If you need more information please ask - I don't know what exactly will be useful to answer my questions.
This article discusses the justification for using western blotting to validate mass spec results. It suggests using selected reaction monitoring assays as an alternative. Some of the points made by commenters in this thread are reiterated in this article.