Rapid Assessment of RNAi-mediated Protein Depletion
by Selected Reaction Monitoring Mass Spectrometry
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Abstract
We describe the use of a targeted
proteomics approach, selected
reaction monitoring (SRM) mass spectrometry, to detect and assess
RNAi-mediated depletion or “knockdown” of specific proteins
from human cells and from <i>Drosophila</i> flies. This
label-free approach does not require any specific reagents to confirm
the depletion of RNAi target protein(s) in unfractionated cell or
whole organism extracts. The protocol described here is general, can
be developed rapidly, and can be multiplexed to detect and measure
multiple proteins at once. Furthermore, the methodology can be extended
to any tandem mass spectrometer, making it widely accessible. This
methodology will be applicable to a wide range of basic science and
clinical questions where RNAi-mediated protein depletion needs to
be verified, or where differences in relative abundance of target
proteins need to be rapidly assessed between samples