RESUMO
Bridging the gap between functional genomics and traditional molecular cell biology is a challenge of the next decade. Here, we are aiming to find routines for targeted quantitation of protein silencing in response to RNAi based on complex cellular lysates. A workflow was established adapting siRNA treatment, processing the sample, generating isobaric iTRAQ-reagent-labeled peptides, and analyzing the sample applying MRM-based peptide quantitation to verify protein silencing on a 4000 QTRAP LC/MS/MS mass spectrometer. Subsequently, eight targets were analyzed, mostly with two siRNA designs. Although transcript and protein silencing correlated, the downregulation on the protein level was less pronounced. A time-course analysis of the chaperon HSPA9/mortalin indicated a delayed kinetic of protein versus transcript silencing. Further, the analysis of the functional response on the example of HSD17B4, a multifunctional enzyme essential to generate precursors for cholesterol biosynthesis, confirmed that strong silencing on the transcript level accompanied by moderate reduction of protein is sufficient to generate a physiological significant response. Fifty percent protein silencing resulted in a 3.5-fold induction of low-density lipoprotein and therefore cholesterol uptake in human liver cells. The established routines pave the way for the development of targeted protein quantitation assays suitable for target and biomarker validation.
