Generating the subcellular human protein atlas using high throughput microscopy R Schutten, H Ait Blal, F Danielsson, M Hjelmare, D Mahdessian, M Skogs, C Stadler, M Wiking, A Åberg, M Uhlén and E Lundberg. Science for Life Laboratory, Royal Institute of Technology (KTH). Tomtebodavägen 23A, 171 65 Solna, Sweden E-mail: [email protected] KEY WORDS: Confocal Microscopy, High throughput, Human Protein Atlas, Human proteome, Subcellular localization. The eukaryotic cell is compartmentalized to ensure that molecules for different processes are concentrated so that those can take place in parallel. Despite much work many questions remain about the spatial organization and the biological function of a large part of the human proteome. Using an antibody-based approach we as part of the Swedish Human Protein Atlas project systematically map the human proteome on a subcellular level [1-3]. Every month 500 proteins are investigated in three cell lines, selected individually for each protein based on RNA expression data from a set of human cell lines. A semi-automated pipeline has been established to combine sample preparation, high-resolution confocal microscopy and image analysis. The high-resolution images obtained enable identification of all major organelles like mitochondria and actin filaments as well as sub-structures like microtubule plus ends and nuclear domains. A strict data validation scheme includes fluorescently tagged proteins (FP), multiple antibodies against the same protein and systematic gene silencing using siRNA [4,5]. More then 10.000 human proteins have so far been localized to one or multiple of the 18 localizations shown in the figure below. Interestingly, these results show that approximately 50% of those proteins are present in multiple locations in the cell and 50% have different locations in different cell lines [2,4]. A lot of information can be gained from the high- resolution images and to harvest this computational image analysis schemes are currently being investigated. To conclude, the aim of the Subcellular Protein Atlas is to provide the first complete view of the localization of all human proteins to enhance our understanding of protein function and cell biology. [1] www.proteinatlas.org/subcellular [2] Fagerberg L et.al. ”Mapping the subcellular protein distribution in three human cell lines,” J Proteome Res. 10(8), 3766-77 (2011). [3] Uhlen M et.al. ”Towards a knowledge-based Human Protein Atlas,” Nat Biotechnol. 28(12), 1248-50 (2010). [4] Stadler C et.al. ”Immunofluorescence and fluorescent-protein tagging show high correlation for protein localization in mammalian cells,” Nat Methods. 10(4), 315-23 (2013). [5] Stadler C et.al. ”Systematic validation of antibody binding and protein subcellular localization using siRNA and confocal microscopy,” J Proteomics. 75(7) 2236–2251 (2012).

Generating the subcellular human protein atlas using high ...M Wiking, A Åberg, M Uhlén and E Lundberg. Science for Life Laboratory, Royal Institute of Technology (KTH). Tomtebodavägen

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Page 1: Generating the subcellular human protein atlas using high ...M Wiking, A Åberg, M Uhlén and E Lundberg. Science for Life Laboratory, Royal Institute of Technology (KTH). Tomtebodavägen

Generating the subcellular human protein atlas using high throughput microscopy

R Schutten, H Ait Blal, F Danielsson, M Hjelmare, D Mahdessian, M Skogs, C Stadler, M Wiking, A Åberg, M Uhlén and E Lundberg.

Science for Life Laboratory, Royal Institute of Technology (KTH). Tomtebodavägen 23A, 171 65 Solna, Sweden

E-mail: [email protected]

KEY WORDS: Confocal Microscopy, High throughput, Human Protein Atlas, Human proteome, Subcellular localization. The eukaryotic cell is compartmentalized to ensure that molecules for different processes are concentrated so that those can take place in parallel. Despite much work many questions remain about the spatial organization and the biological function of a large part of the human proteome. Using an antibody-based approach we as part of the Swedish Human Protein Atlas project systematically map the human proteome on a subcellular level [1-3]. Every month 500 proteins are investigated in three cell lines, selected individually for each protein based on RNA expression data from a set of human cell lines. A semi-automated pipeline has been established to combine sample preparation, high-resolution confocal microscopy and image analysis. The high-resolution images obtained enable identification of all major organelles like mitochondria and actin filaments as well as sub-structures like microtubule plus ends and nuclear domains. A strict data validation scheme includes fluorescently tagged proteins (FP), multiple antibodies against the same protein and systematic gene silencing using siRNA [4,5]. More then 10.000 human proteins have so far been localized to one or multiple of the 18 localizations shown in the figure below. Interestingly, these results show that approximately 50% of those proteins are present in multiple locations in the cell and 50% have different locations in different cell lines [2,4]. A lot of information can be gained from the high-resolution images and to harvest this computational image analysis schemes are currently being investigated. To conclude, the aim of the Subcellular Protein Atlas is to provide the first complete view of the localization of all human proteins to enhance our understanding of protein function and cell biology.

[1] www.proteinatlas.org/subcellular [2] Fagerberg L et.al. ”Mapping the subcellular protein distribution in three human cell lines,” J Proteome Res. 10(8), 3766-77 (2011). [3] Uhlen M et.al. ”Towards a knowledge-based Human Protein Atlas,” Nat Biotechnol. 28(12), 1248-50 (2010). [4] Stadler C et.al. ”Immunofluorescence and fluorescent-protein tagging show high correlation for protein localization in mammalian cells,” Nat Methods. 10(4), 315-23 (2013). [5] Stadler C et.al. ”Systematic validation of antibody binding and protein subcellular localization using siRNA and confocal microscopy,” J Proteomics. 75(7) 2236–2251 (2012).