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Systems Biology — the Broader Perspective
Open AccessConcept Paper

Quantification of High-Molecular Weight Protein Platforms by AQUA Mass Spectrometry as Exemplified for the CD95 Death-Inducing Signaling Complex (DISC)

1
Functional Proteome Analysis, German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany
2
Division of Immunogenetics, German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany
3
Department of Translational Inflammation Research, Institute of Experimental Internal Medicine, Otto von Guericke University, 39120 Magdeburg, Germany
*
Author to whom correspondence should be addressed.
These authors contributed equally to this work.
Cells 2013, 2(3), 476-495; https://doi.org/10.3390/cells2030476
Received: 8 May 2013 / Revised: 29 May 2013 / Accepted: 19 June 2013 / Published: 27 June 2013
(This article belongs to the Special Issue Successes of Systems Biology and Future Challenges)
Contemporary quantitative mass spectrometry provides fascinating opportunities in defining the stoichiometry of high-molecular weight complexes or multiprotein platforms. The composition stoichiometry of multiprotein platforms is a key to understand the regulation of complex signaling pathways and provides a basis for constructing models in systems biology. Here we present an improved AQUA technique workflow that we adapted for the quantitative mass spectrometry analysis of the stoichiometry of the CD95 (Fas/APO-1) death inducing signaling complex (DISC). The DISC is a high-molecular weight platform essential for the initiation of CD95-mediated apoptotic and non-apoptotic responses. For protein quantification, CD95 DISCs were immunoprecipitated and proteins in the immunoprecipitations were separated by one-dimensional gel electrophoresis, followed by protein quantification using the AQUA technique. We will discuss in detail AQUA analysis of the CD95 DISC focusing on the key issues of this methodology, i.e., selection and validation of AQUA peptides. The application of this powerful method allowed getting new insights into mechanisms of procaspase-8 activation at the DISC and apoptosis initiation [1]. Here we discuss the AQUA methodology adapted by us for the analysis of the CD95 DISC in more detail. This approach paves the way for the successful quantification of multiprotein complexes and thereby delineating the intrinsic details of molecular interactions. View Full-Text
Keywords: CD95; DISC; apoptosis; DED; protein quantification; mass spectrometry; AQUA technique; protein platforms CD95; DISC; apoptosis; DED; protein quantification; mass spectrometry; AQUA technique; protein platforms
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Warnken, U.; Schleich, K.; Schnölzer, M.; Lavrik, I. Quantification of High-Molecular Weight Protein Platforms by AQUA Mass Spectrometry as Exemplified for the CD95 Death-Inducing Signaling Complex (DISC). Cells 2013, 2, 476-495.

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