Interaction Proteomics

Proteins do not exert their function by random diffusion as single entities in cells but in the form of molecular machines, composed of several different proteins or multiple copies of a single protein. Each protein complex is an assembly of proteins, more or less stably associated or interacting in a dynamic fashion. The assembly of proteins into complexes results in a limited number of possible reaction partners and thereby restricts the number of potential reactions. Additionally, the organization of the complex leads to a sequential order of reactions and thus creates a highly structured and regulated reaction space. The modular use of proteins within dynamically regulated protein complexes increases systemic flexibility providing fast adaption to the needs of cells in a certain state or environment.

With the aim to circumvent inherent limitations of existing methods for the analysis of protein complexes we develop, optimize and apply analytical workflows for the purification and identification of protein complexes like the SF-TAP (Gloeckner et al, Proteomics 2007; Gloeckner et al, Curr Protoc Protein Sci 2009; Gloeckner et al, Methods Mol Biol 2009; Boldt et al, Methods Cell Biol 2009) or the QUICK approach (Meixner et al, MCP 2011; Analysis of LRRK2 protein complexes) which was originally developed by Selbach and Mann. We have demonstrated the usability of these methods for various targets (den Hollander et al, Nature Genetics 2007; Kinzel and Boldt et al, Dev Cell 2010) and are now using the SF-TAP approach for large scale mapping of the ciliary protein network in a systems biology approach (http://www.syscilia.org/).

Currently we are extending our repertoire to quantitative methods for detecting protein complexes in native tissue as well as for the quantitative and comparative analysis of protein complexes by mass spectrometry. These approaches will not only enable us to identify protein complexes with high confidence from cells and tissues but also to detect alterations within the complexes, induced by perturbations like mutations and exogenic noxes.



A) Strep/FLAG-TAP overview I: Purification by the tandem Strep-tag II moiety: Binding to Strep-Tactin matrix (upper panel), elution with desthiobiotin (lower panel). II: Purification by the FLAG-tag moiety: binding to anti FLAG M2 affinity matrix (upper panel), elution with FLAG peptide (lower panel).
B) Quantitative protein complex comparison overview: Protein complexes are purified from SILAC labelled cells, combined before mass spectrometric analysis and quantified by Max Quant. The identified alterations within the complex are visualized as protein network. Increased binding is shown as green node, decreased binding as red node.
A) and B) Specific interactors are shown as yellow circles, non-specific proteins (contaminants) are shown as light blue circles.

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