RNAi and iTRAQ reagents united: targeted quantitation of siRNA-mediated protein silencing in human cells.
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.
Abdrakhmanova
A
A
Applied Biosystems, Life Technologies, Frankfurter Strasse 129B, 64293, Darmstadt, Germany.
Schlichting
R
R
Hunter
C L
CL
Glueckmann
M
M
Lenz
C
C
Echeverri
C J
CJ
Soennichsen
B
B
Jung
A
A
Weiss-Haljiti
C
C
D015067:17-Hydroxysteroid Dehydrogenases / Q000032:analysis* / Q000235:genetics; D000595:Amino Acid Sequence; D045744:Cell Line, Tumor; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000032:analysis* / Q000235:genetics; D018840:HSP70 Heat-Shock Proteins / Q000032:analysis* / Q000235:genetics; D006801:Humans; D006836:Hydro-Lyases / Q000032:analysis* / Q000235:genetics; D008077:Lipoproteins, LDL / Q000378:metabolism; D008099:Liver / Q000166:cytology; D024101:Mitochondrial Proteins; D008969:Molecular Sequence Data; D064029:Peroxisomal Multifunctional Protein-2; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics*; D053719:Tandem Mass Spectrometry
D018840:HSP70 Heat-Shock Proteins; C492001:HSPA9 protein, human; D008077:Lipoproteins, LDL; D024101:Mitochondrial Proteins; D034741:RNA, Small Interfering; C080352:mortalin; D015067:17-Hydroxysteroid Dehydrogenases; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D006836:Hydro-Lyases; D064029:Peroxisomal Multifunctional Protein-2; C104587:HSD17B4 protein, human