I am working on TMT-labelled proteomics data (3 conditions in triplicates). Looking at the mean-variance relationship of my data on the peptide-level, it appears to be in good agreement with an additive-multiplicative error model, so I used vsn2 to transform the data. Now that the variance is stabilized, I believe I could directly use limma to asses the significance of changes on the peptide level. However, I would like to have a model for the significance on the protein level, which takes into account the number of detected peptides per protein (which is quite variable) and assigns higher significance to proteins with several peptides showing consistent changes than with just a single peptide showing the same change. Is there a way to use limma (on the vsn-transformed data) to this end?
I can't speak for what happens with proteomics data, but in general, a variance-stabilizing normalization is not a prerequisite for analyses with
limma
. Instead, you can model the mean-variance relationship by runningeBayes
withtrend=TRUE
.Edit: To be clear, I'm referring to the VSN procedure done by
method="vsn"
. Most analyses start off with log-transformed intensities, which already stabilizes the variance a bit. My point is that we usually don't bother with more sophisticated stabilization procedures, and trustlimma
(orvoom
, for RNA-seq) to handle the modelling of the mean-variance relationship.Aaron - that's a surprising statement, chapters 6 and 8 of the limma users guide recommend log-transformation and background correction, which together have an approximate variance-stabilising effect. Are you saying one shouldn't do this and just feed untransformed intensities into limma? (for microarrays, or any other technology)
You're right, my apologies; I was referring specifically to the
method="vsn"
option innormalizeBetweenArrays
. Comment's been amended.