Since most cellular processes depend on interactions between proteins, information about protein-protein interactions (PPIs) provide valuable insights into protein function. Over the last years, quantitative affinity purification followed by mass spectrometry (q-AP-MS) has become a powerful approach to investigate PPIs in an unbiased manner. In q-AP-MS the protein of interest is biochemically enriched together with its interaction partners. In parallel, a control experiment is performed to control for non-specific binding. Quantitative mass spectrometry is then employed to compare protein levels in both samples and to exclude non-specific contaminants. Here, we provide two detailed q-AP-MS protocols for pull-downs with immobilized bait proteins or transient transfection of tagged expression constructs. We discuss benefits and limitations of q-AP-MS and highlight critical parameters that need to be considered. The protocols and background information presented here allow the reader to adapt the generic q-AP-MS strategy for a wide range of biological questions.
"Proteins often functionally interact as part of complex and dynamic networks  . An important goal of current proteomics research is to develop techniques that can accurately identify such protein 'interactomes' . A popular method uses epitope-tagged proteins in pull-down assays. "
"We performed immuno-based affinity purification experiments followed by mass spectrometric protein identification (46) using the E13-inducible ES clones with the 3xFLAG (‘Materials and Methods’ section). We identified 23 potential protein partners of E13 with high-confidence (Table 2), among which there are two TFs [Gtf2e2, Btf3 (47)], several mRNA processing proteins and two components of the Polycomb complex [Eed, Suz12 (48) and the retinoblastoma binding protein Rbbp4, which may be involved in pluripotent stem cell maintenance and neuronal differentiation (49)]. "
[Show abstract][Hide abstract] ABSTRACT: Gene expression profiles can be used to infer previously unknown transcriptional regulatory interaction among thousands of genes, via systems biology 'reverse engineering' approaches. We 'reverse engineered' an embryonic stem (ES)-specific transcriptional network from 171 gene expression profiles, measured in ES cells, to identify master regulators of gene expression ('hubs'). We discovered that E130012A19Rik (E13), highly expressed in mouse ES cells as compared with differentiated cells, was a central 'hub' of the network. We demonstrated that E13 is a protein-coding gene implicated in regulating the commitment towards the different neuronal subtypes and glia cells. The overexpression and knock-down of E13 in ES cell lines, undergoing differentiation into neurons and glia cells, caused a strong up-regulation of the glutamatergic neurons marker Vglut2 and a strong down-regulation of the GABAergic neurons marker GAD65 and of the radial glia marker Blbp. We confirmed E13 expression in the cerebral cortex of adult mice and during development. By immuno-based affinity purification, we characterized protein partners of E13, involved in the Polycomb complex. Our results suggest a role of E13 in regulating the division between glutamatergic projection neurons and GABAergic interneurons and glia cells possibly by epigenetic-mediated transcriptional regulation.
Nucleic Acids Research 11/2012; 41(2). DOI:10.1093/nar/gks1136 · 9.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: High-throughput genomic sequencing and quantitative mass spectrometry (MS)-based proteomics technology have recently emerged as powerful tools, increasing our understanding of chromatin structure and function. Both of these approaches require substantial investments and expertise in terms of instrumentation, experimental methodology, bioinformatics, and data interpretation and are, therefore, usually applied independently from each other by dedicated research groups. However, when applied reiteratively in the context of epigenetics research these approaches are strongly synergistic in nature.
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