"Tissue- and isoform-specific protein...


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

Proteins are associated with specific molecular functions in the cells, which are comprised by protein-complexes rather than by single proteins. The assembly of these complexes is tissue and cell type specific and therefore the analysis of these complexes needs to be performed or at least validated in relevant tissues. To achieve this Beyer et al. established a fast and efficient approach to investigate tissue-specific protein complexes and demonstrate that this can indeed be used to detect tissue specific interactions for example from retina or pigment epithelial cells.

Graphical abstract: The protein of interest was produced in a high amount by overexpression in HEK293T cell culture. As control, an unrelated protein was used. All proteins were fused to a FLAG-tag. For bait purification, FLAG-tagged proteins were immobilized using FLAG antibody bound to agarose beads. HEK293T based carry-over was removed by mild washing using low SDS concentrations. The target tissue (here shown pig retina) was prepared and added to the bead-bound purified protein of interest. After incubation, non-bound proteins were removed and proteins bound to the beads were eluted. The protein mixture was analyzed by mass spectrometry, identification, quantification and statistical analyses were performed by using MaxQuant and Perseus.

Protein-protein interaction analysis is widely used to identify the molecular function of proteins and their connectivity within protein networks. To facilitate the investigation of protein interactions, recombinant expression of a given protein of interest in cell lines yielding high protein expression levels is frequently used. Performing protein-protein interaction analyses in cultured cells has several advantages, including easy handling, inexpensive cell and protein propagation and fast genetic manipulation. For these reasons, cell culture-based protein complex analysis has proven to be helpful to get first insights into protein complex formation and mutation-specific changes. On the other hand, protein function is context-dependent and assembly as well as activity of protein complexes depends on tissue-specific regulation of gene expression. This becomes apparent when mutations in ubiquitously expressed genes result in diseases that only affect specific tissues or organs.

So far, immunoprecipitation of proteins and protein complexes from tissues or organs has been the method of choice to gain insights into tissue- and context-specific physiological interactions of proteins. Here, specific antibodies for each protein of interest with high affinity to their target protein have to be established, which is time-consuming and expensive. Recently, gene editing techniques depending on CRISPR/Cas have been established to insert a tag sequence into the genome resulting in endogenous expression of a tag-fusion protein. When used to genetically engineer transgenic model animals, low expression levels as well as the high amount of material required may be limiting. Moreover, these methods are inefficient, time-consuming and not applicable to the analysis of human intact tissue. To overcome the above-mentioned limitations and to provide a fast and at the same time- and cost-efficient approach, an approach was developed that combines the advantages of standardized mammalian cell culture for bait production and enrichment with its application to analyze protein interactions from different organs and tissues. Tissue-specific interactions were reproducibly identified from porcine retina as well as from retinal pigment epithelium using the ciliary protein lebercilin as bait. Further, murine heart-specific interactors of two gene products of the 3′,5′-cyclic guanosine monophosphate (cGMP)-dependent protein kinase type 1 (cGK1) were investigated. Here, specific interactions were associated with the cGK1α and β gene products, that differ only in their unique amino-terminal region comprising about 100 aa. As such, the new protocol provides a fast and reliable method for tissue-specific protein complex analysis which is independent of the availability or suitability of antibodies for immunoprecipitation.

Publication link: https://www.sciencedirect.com/science/article/abs/pii/S1874391920303158?via%3Dihub

Beyer T, Klose F, Kuret A, Hoffmann F, Lukowski R, Ueffing M, Boldt K. Tissue- and isoform-specific protein complex analysis with natively processed bait proteins. J Proteomics. 2020 Aug 24:103947. doi: 10.1016/j.jprot.2020.103947. Epub ahead of print. PMID: 32853754.