Pseudotargeted MS Method for the Sensitive Analysis of Protein Phosphorylation in Protein Complexes

In this study, we
presented an enrichment-free approach for the
sensitive analysis of protein phosphorylation in minute amounts of
samples, such as purified protein complexes. This method takes advantage
of the high sensitivity of parallel reaction monitoring (PRM). Specifically,
low confident phosphopeptides identified from the data-dependent acquisition
(DDA) data set were used to build a pseudotargeted list for PRM analysis
to allow the identification of additional phosphopeptides with high
confidence. The development of this targeted approach is very easy
as the same sample and the same LC-system were used for the discovery
and the targeted analysis phases. No sample fractionation or enrichment
was required for the discovery phase which allowed this method to
analyze minute amount of sample. We applied this pseudotargeted MS
method to quantitatively examine phosphopeptides in affinity purified
endogenous Shc1 protein complexes at four temporal stages of EGF signaling
and identified 82 phospho-sites. To our knowledge, this is the highest
number of phospho-sites identified from the protein complexes. This
pseudotargeted MS method is highly sensitive in the identification
of low abundance phosphopeptides and could be a powerful tool to study
phosphorylation-regulated assembly of protein complex.