- The amount of spiked-in volume should be discussed with the
scientist performing the mass spectrometry experiments. - After receiving the dataset, normalize the “light” peptide
intensity data points by normalization factors obtained from
“heavy” peptide intensity. Linear regression between the
“heavy” intensity from both conditions may be applied and
the slope of the linear fit used as normalization factor. Alterna-
tively, normalization may also be carried out by quantile nor-
malization (preprocessCore, baySeq Bioconductor packages)
[25] or DESeq2-style normalization which is based on geo-
metric means [26]. Identify differential binders by computing
log2-transformed fold changes between treated and untreated
sample. - Differential amount of proteins in oligo(dT) eluates may arise
due to differences in amounts of total poly(A)+RNA between
treated and untreated cells as well as differences in total protein
abundance. Therefore, mRNA and protein levels should be
quantified in input lysates by qRT-PCR, RNA-seq, Western,
and whole proteome analysis. If changes can only be observed
on the RBPome level, but not on the mRNA and protein level
the differential binding activity for RNA-binding proteins was
successfully detected.
Acknowledgment
We thank Koshi Imami and Matthias Selbach (Max Delbr€uck Cen-
ter for Molecular Medicine, Berlin) for their expertise in mass
spectrometry. This work was supported by an International Euro-
pean Fellowship (Maria Sklodowska Actions FP7-PEOPLE-2011-
IEF) and DFG grant LA 2941/5-1.References
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416 Miha Milek and Markus Landthaler