Dataset Information

Tissue-specific protein complex analysis with natively processed bait proteins

ABSTRACT: Protein-protein interaction analysis is an important tool to elucidate the function of proteins and protein complexes as well as their dynamic behavior. Despite the large toolset available to date the analysis of tissue specific complexes is still relying on the availability of specific antibodies suited for immunoprecipitation. Due to the lack of specific, high affinity antibodies, we aimed at establishing a novel approach that is generally applicable to identify protein interactions and complexes in tissue. Therefore, we established an approach for which a tagged bait protein is expressed in a mammalian cell line which is, after a specific cleaning procedure, used to pulldown prey proteins and complexes from tissue samples. We demonstrate here that this protocol allows the identification of tissue specific interaction by applying it to the ciliary protein lebercilin and its specific interactors were identified in retina or the retinal pigment epithelium. Further, the heart specific interactome of two isoforms of the cyclic guanosine-3’,5’-monophosphate (cGMP)-dependent protein kinase type 1α and 1β (cGKI/ aka PRKG1/), which differ only in their unique amino-terminal region comprising about 100 AS, shows the suitability of the approach to identify isoform-specific interactions. With this affinity purification-based protocol, we provide a fast and reliable method for tissue-specific protein complex analysis independent of antibody availability.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Sus Scrofa Domesticus (domestic Pig)

TISSUE(S): Heart

SUBMITTER: Tina Beyer

LAB HEAD: Marius Ueffing

PROVIDER: PXD018047 | Pride | 2023-12-28

REPOSITORIES: Pride

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Publications

Tissue- and isoform-specific protein complex analysis with natively processed bait proteins.

Beyer Tina T Klose Franziska F Kuret Anna A Hoffmann Felix F Lukowski Robert R Ueffing Marius M Boldt Karsten K

Journal of proteomics 20200824

Protein-protein interaction analysis is an important tool to elucidate the function of proteins and protein complexes as well as their dynamic behavior. To date, the analysis of tissue- or even cell- or compartment-specific protein interactions is still relying on the availability of specific antibodies suited for immunoprecipitation. Here, we aimed at establishing a method that allows identification of protein interactions and complexes from intact tissues independent of specific, high affinity ...[more]

PMID: 32853754

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Project description:To discover RBPs with increased insolubility in a human ALS model, we applied a well established dual-SMAD inhibition-based protocol (Fang et al., 2019; Markmiller et al., 2021; Markmiller et al., 2018; Martinez et al., 2016) to generate iPSC-MN from six control iPSC lines, from four iPSC lines originating from two sALS patients, and from two iPSC lines originating from fALS patients with pathogenic variants in the TARDBP gene (Table S1; Figure S1A). No difference in differentiation capacity was observed (Figure S1B-G), resulting in average 40% ISL1+ MN (Figures S1G), comparable to numbers observed in large scale MN differentation studies (Baxi et al., 2022). The susceptibility of ALS MN to sodium arsenite-induced stress was not changed (Figure S1H and I). Next, we asked which proteins exhibit an increased insolubility in our ALS iPSC-MN. We fractioned iPSC- MN by lysis in radio-immunoprecipitation assay (RIPA) buffer, followed by ultracentrifugation and solubilization of RIPA insoluble proteins in urea buffer. The ultracentrifugation-cleared RIPA insoluble protein fraction is widely used to study protein insolubility in the context of neurodegeneration (Nuber et al., 2013; Walker et al., 2015). Label-free mass spectrometry of the insoluble protein fraction was utilized to identify proteins that are insoluble in sALS and fALS, relative to control iPSC-MNs (Figure 1A). Gene ontology (GO) analysis of the 100 proteins (top 2.9% of all detected proteins) with the highest label free quantification (LFQ) intensities in controls (Figure S1J) revealed that ‘unfolded protein binding’ (corrected P = 7.95 x 10-16) and ‘structural constituent of cytoskeleton’ (corrected P = 1.47 x 10-10) were among the 10 most significantly enriched GO terms, indicating enrichment of insoluble proteins (Figure S1K). Principle component analysis of the insoluble fractions did not distinguish ALS from control samples, suggesting that the overall insoluble proteome is not changed (Figure S1L). At threshold P ≤ 0.05 (Welch’s t-test) and fold change ≥ 1.5, we identified 88 proteins enriched in the insoluble fraction in ALS samples relative to control (Figure 1B). When the sample labels were randomly shuffled, we observed an average of 7.5 proteins (~12-fold lower) as differentially enriched at the same statistical thresholds, indicative of an ALS-specific protein insolubility pattern (Figure 1C). The 88 candidate proteins included cytoskeletal components and motor proteins, functional categories associated with prominent ALS in vitro phenotypes (Akiyama et al., 2019; Egawa et al., 2012; Fazal et al., 2021; Guo et al., 2017; Kreiter et al., 2018) (Figure 1D). Notably, 5 RBPs, NOVA1, ELAVL4, FXR2, RBFOX2, and RBFOX3 were also enriched (Figure 1D). The NOVA1 paralog NOVA2 was significantly enriched (P = 0.03) but did not meet our enrichment threshold (fold change = 1.36). Interestingly, insoluble TDP-43 protein was not significantly different in ALS and control (P = 0.98; fold change = 0.97). Western blot analysis confirmed the increase in insolubility of NOVA1, NOVA2, ELAVL4, RBFOX2 and RBFOX3 (Figure 1E and 1F). The soluble protein levels of NOVA1 and NOVA2 were also increased (Figure 1F). In conclusion, we identified 5 RBPs with elevated insoluble protein levels of ALS-iPSC-MNs.

Dataset Information

Tissue-specific protein complex analysis with natively processed bait proteins

Publications

Tissue- and isoform-specific protein complex analysis with natively processed bait proteins.

Similar Datasets

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