APROXIMACIÓN A LOS RESULTADOS OBTENIDOS POR EL USO DE LAS RENTAS GENERADAS POR LA ACTIVIDAD

The evaluation of different lysis conditions as well as the assessment of various immunoprecipitation and affinity purification methods followed by the final decision for the most promising strategy represents one of the most crucial parts in interactome analyses. From mass spectrometry based assays emerge analytical challenges relative to critical assessment of potentially identified interactors in furtherance of optimizing experimental conditions, while achieving an unbiased screening method revealing a reliable dataset. To that end, several parameters have to be considered for the unbiased evaluation. The first and probably most important issue one has to overcome is a sufficient enrichment level of the bait protein. The presence of an appropriate number of peptide identifications is crucial for the successful identification of interaction partners, as a considerable number of pulled down bait protein increases the probability of detecting endogenously low expressed interacting proteins. Furthermore, an appropriate dataset of identified co-purified proteins; and in case of quantitative approaches, the number of quantified proteins; are essential for the evaluation of different experimental and analytical parameters. Therefore, careful planning provides the possibility to unravel suboptimal experimental conditions that inhibit binding capacities, likewise inappropriate buffer conditions interfering with the purification system. Another important step in the assessment procedure of interactome analysis is the reproducibility of generated interaction data. There has to be an obvious overlap in interacting proteins when performing multiple biological and technical replicates. This is not only a matter of suitable purification methods or exact implementation of the workflow for each experiment, but can also be induced by the susceptibility of the highly sensitive mass spectrometers towards environmental contaminants and/or carryover of residual contaminations from previous runs. For interactome analysis, the expression level of the protein of interest could vary dramatically with transient transfection efficiency. Whereas, the endogenous expression is closer to the cell physiological conditions, this approach raises several challenges. First, one has to find a suitable cell line that expresses the protein on a certain level and second, co-immunoprecipitation of endogenous proteins requires

131 the availability of applicable antibodies. For the transcription factor SOX11, which we chose as bait for the interactome study, the selected neuroblastoma cell line Neuro2a exhibited a basal protein expression. Other, probably more beneficial cell lines for the investigation of neurogenesis-related regulatory processes like neuronal precursor cells isolated from neurogenic niches of adult mouse brains were excluded due to the dramatic loss of SOX11 expression after several passages in culture. Additionally, these cells present limitation with sufficient expansion to gain a suitable amount of nuclear lysates, which was required for an efficient immunoprecipitation assay. Commercially available anti-SOX11 antibodies were classified as not suitable for the pull down experiments due to inefficient enrichment of SOX11 combined with changing performance of polyclonal antibodies from different charges and high costs for the application in multiple experiments. However, one group performed in 2012 co-immunoprecipitations using a commercial anti-Sox11 purchased from Santa Cruz for pull down assays in mouse embryonic kidney cells on endogenous level. Hence, they were able to validate the interaction of SOX11 with WT1 (Wilm’s tumor suppressor gene 1), a factor that regulates the WNT4 gene, which is involved in nephrogenesis together with SOX11 in a synergistic fashion (Murugan et al., 2012). This could result from higher SOX11 expression levels in the applied cell system compared to the Neuro2a cells. Moreover, they only investigated the co-precipitation of one protein, therefore no complete interactome was determined. In this present study, the applied strategy to overcome the lack of appropriate anti-SOX11 antibodies was the production of peptide- as well as protein-based monoclonal antibodies in house using a SOX11-specific protein recombinant epitope signature tag (PrEST) for immunisation. It represents a unique SOX11 specific fragment comprised of 117 amino acids, designed by an antigen design software, which is not part of transmembrane regions or signal peptides (Uhlen et al., 2010). Extensive screenings revealed several antibodies suitable for western blot analysis, as illustrated in figures 17-19. However, they were not applicable in co- immunoprecipitation assays, displayed in figure 20. This effect could be caused by an impaired binding capacity of the antibodies to the natively folded protein compared to the denatured transcription factor in western blotting. Additionally, the basal endogenous expression level of SOX11 may not provide sufficient material for an efficient precipitation. Finally, the endogenous co-immunoprecipitation of SOX11 was not realisable given the circumstances. On that account, affinity purifications

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were selected as the method of choice for the interactome screen. This strategy enables the use of tagged SOX11 constructs and thus circumvents the need for specific antibodies. A combined Strep FLAG tag (SF-TAP tag), comprised of a tandem Strep II and one single FLAG tag, suitable for highly efficient Strep FLAG tandem affinity purification (SF-TAP) as published before (Gloeckner et al., 2007) was fused to the N-terminus of the recombinant SOX11 expression construct via Gateway Cloning. Both parts of the two-step affinity purification strategy are also suitable for the performance as one-step purifications. The quantitative mass spectrometric approach applied for SOX11 interactome analysis using SILAC labelling was based on ratios quantification by comparing identified peptides of the experimental versus the control condition, leading to the discrimination of specific or unspecific identified binding partners. Consequently, a certain number of unspecific signals was required for a reliable interpretation of performed affinity purifications. As for the single-step Strep purification method in general, a relative high number of unspecific binding partners compared to the more pure FLAG affinity purifications was observed, this strategy was initially applied for the implementation of the SOX11 interactome. However, the presence of high salt concentrations in the nuclear extraction buffer C, which is needed to extract proteins efficiently from the nucleus, appeared to interfere with the buffer system of the Strep purification and inhibited binding of SOX11 to the Strep-Tactin matrix, as illustrated in figure 21. Reducing the NaCl concentration from 420mM to 210mM in the nuclear lysate preparation workflow led to a recovered binding capacity of the Strep II tag to the beads, but at the same time the quality of the obtained interaction partner list was not satisfying. This brought up the assumption of an incomplete extraction of nuclear proteins caused by the decreased salt concentration. The Strep purification method is suitable for the pull down of proteins from bacteria lysates as well as eukaryotic cytoplasmic proteins (Schmidt and Skerra, 2007), but it was not applied in many studies working with nuclear lysates, although one group claimed the robustness of Strep II–mediated purifications up to a NaCl concentration of 400mM in the reaction buffer concerning the efficiency of bait protein precipitation (Junttila et al., 2005). However, they did not mention the yield of interaction partner pull down, that were for sure much less expressed in relation to the protein of interest and were potentially not extracted to the same extend as usual under the high salt conditions. Another study implicating

133 the determination of the SOX2 interactome in embryonic stem cells reported the application of Strep pull down assays for the validation of interactors identified by FLAG affinity purification on western blot analysis (Gao et al., 2012). Here, the nuclear extraction was carried out using a kit where the salt concentration was not indicated. Furthermore, the Strep purification was performed in reaction buffers comprising solely 150mM NaCl. More frequently, FLAG affinity purifications were used for the analysis of nuclear protein complexes, like in an interactor screen of SOX2 in murine neural stem cells stably expressing SOX2 (Engelen et al., 2011). Consequently the FLAG one-step purification was applied for the Sox11 interactome analysis, where best performance was observed with the original nuclear extraction buffer conditions, comprising 420mM NaCl, as displayed in figure 22. In order to be closer to the physiological SOX11 level, stable SOX11 expression cell lines were included in the purification procedure, however the small number of detected peptides specific for SOX11 due to the low expression state rendered an interactome screen impossible. On that account, the FLAG pull down assays were conducted using transiently over-expressed SF-TAP tagged SOX11. The subsequent Methanol/Chloroform precipitation followed by in-solution tryptic digestion turned out to be the optimal sample preparation, as by the use of in-gel pre-fractionation peptide identification rates for the bait as well as for the interacting proteins decreased. This illustrated that the sample was rather pure than complex, which rendered a pre- separation unnecessary, as every redundant step results in sample loss. The implementation of 6 independent experiments, thereof 3 including reverse labelling and subsequent data analysis revealed a list of interactors either considered as specific or non-specific. The obtained high purity of the performed FLAG affinity purifications due to the high affinity of the FLAG tag to the matrix compared to the Strep tag (Gloeckner et al., 2007) led to a not exactly Gaussian distribution of the data, which were shifted towards the SOX11 condition, as illustrated in figure 22. That implies that the low abundance of unspecific binders which were needed to a certain extend for the quantification of specific signals and ratios calculations led to the assignment of genuine SOX11 interaction partners as not significantly enriched. To overcome this problem of data evaluation, a threshold was set manually, defining all proteins as significant interaction partners of SOX11 that revealed SOX11/control ratios above a value of 2.

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5.2. Sox11 interactors are involved in transcriptional regulation and