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RNAi and iTRAQ reagents united: targeted quantitation of siRNA-mediated protein silencing in human cells

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Abstract

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.

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Abbreviations

RNAi:

RNA interference

siRNA:

Short interfering RNA

iTRAQ®:

Isotope tags for relative and absolute quantification

MRM:

Multiple-reaction monitoring

MS:

Mass spectrometry

qRT-PCR:

Quantitative real-time-PCR

WB:

Western blot

GAPDH:

Glyceraldehyde 3-phosphate dehydrogenase

miRNA:

micro RNA

LDL:

Low-density lipoprotein

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Acknowledgments

We thank Kathleen Boerner, Sanna Olsson, and Nicole Kiessling for their technical assistance and Andrew Walsh for bioinformatic support. We thank Marjorie S. Minkoff and Michael Hannus for technical advice setting up sample preparation and using the LDL-DiI uptake assay, respectively. Part of this work was supported by grant no. 0313815A and 0313815-C of the BMBF.

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Authors and Affiliations

  1. Applied Biosystems, Life Technologies, Frankfurter Straße 129B, 64293, Darmstadt, Germany

    A. Abdrakhmanova, M. Glueckmann, C. Lenz & A. Jung

  2. Applied Biosystems, Life Technologies, 850 Lincoln Centre Drive, Foster City, 94404, CA, USA

    C. L. Hunter

  3. Cenix BioScience GmbH, Tatzberg 47, 01307, Dresden, Germany

    A. Abdrakhmanova, R. Schlichting, C. J. Echeverri, B. Soennichsen & C. Weiss-Haljiti

  4. Nycomed GmbH, Byk-Gulden-Str. 2, 78467, Konstanz, Germany

    C. Weiss-Haljiti

  5. Knauer GmbH, Hegauer Weg 38, 14163, Berlin, Germany

    A. Abdrakhmanova

  6. Boehringer Ingelheim Pharma GmbH, Birkendorfer Str. 65, 88400, Biberach (Riss), Germany

    A. Jung

Authors
  1. A. Abdrakhmanova
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  2. R. Schlichting
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  3. C. L. Hunter
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  4. M. Glueckmann
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  5. C. Lenz
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  6. C. J. Echeverri
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  7. B. Soennichsen
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  8. A. Jung
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  9. C. Weiss-Haljiti
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Corresponding author

Correspondence to C. Weiss-Haljiti.

Additional information

A. Abdrakhmanova and R. Schlichting contributed equally to this work.

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Abdrakhmanova, A., Schlichting, R., Hunter, C.L. et al. RNAi and iTRAQ reagents united: targeted quantitation of siRNA-mediated protein silencing in human cells. Anal Bioanal Chem 395, 773–785 (2009). https://doi.org/10.1007/s00216-009-3028-x

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  • DOI: https://doi.org/10.1007/s00216-009-3028-x

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