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1. A subcellular map of the human proteome

Author

Thul, Peter J., Åkesson, Lovisa, Wiking, Mikaela, Mahdessian, Diana, Geladaki, Aikaterini, Blal, Hammou Ait, Alm, Tove, Asplund, Anna, Björk, Lars, Breckels, Lisa M., Bäckström, Anna, Danielsson, Frida, Fagerberg, Linn, Fall, Jenny, Gatto, Laurent, Gnann, Christian, Hober, Sophia, Hjelmare, Martin, Johansson, Fredric, Lee, Sunjae, Lindskog, Cecilia, Mulder, Jan, Mulvey, Claire M., Nilsson, Peter, Oksvold, Per, Rockberg, Johan, Schutten, Rutger, Schwenk, Jochen M., Sivertsson, Åsa, Sjöstedt, Evelina, Skogs, Marie, Stadler, Charlotte, Sullivan, Devin P., Tegel, Hanna, Winsnes, Casper, Zhang, Cheng, Zwahlen, Martin, Mardinoglu, Adil, Pontén, Fredrik, von Feilitzen, Kalle, Lilley, Kathryn S., Uhlén, Mathias, and Lundberg, Emma

Published
2017

2. Analysis of the Human Tissue-specific Expression by Genome-wide Integration of Transcriptomics and Antibody-based Proteomics

Author

Fagerberg, Linn, Hallström, Björn M., Oksvold, Per, Kampf, Caroline, Djureinovic, Dijana, Odeberg, Jacob, Habuka, Masato, Tahmasebpoor, Simin, Danielsson, Angelika, Edlund, Karolina, Asplund, Anna, Sjöstedt, Evelina, Lundberg, Emma, Szigyarto, Cristina Al-Khalili, Skogs, Marie, Takanen, Jenny Ottosson, Berling, Holger, Tegel, Hanna, Mulder, Jan, Nilsson, Peter, Schwenk, Jochen M., Lindskog, Cecilia, Danielsson, Frida, Mardinoglu, Adil, Sivertsson, Åsa, von Feilitzen, Kalle, Forsberg, Mattias, Zwahlen, Martin, Olsson, IngMarie, Navani, Sanjay, Huss, Mikael, Nielsen, Jens, Ponten, Fredrik, and Uhlén, Mathias

Published
2014
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7. Antibody-based subcellular localization of the human proteome

Author

Skogs, Marie

Subjects
Organelles, Subcellular localization, Permeabilization, Cellbiologi, Immunofluorescence, Antibody validation, Human proteome, Cell Biology, Fixation
Abstract

This thesis describes the use of antibodies and immunofluorescence for subcellular localization of proteins. The key objective is the creation of an open-source atlas with information on the subcellular location of every human protein. Knowledge of the spatial distribution and the precise location of a protein within a cell is important for its functional characterization, and describing the human proteome in terms of compartment proteomes is important to decipher cellular organization and function. Immunofluorescence and confocal microscopy of cultured cells were used for high-resolution detection of proteins on a high-throughput scale. Critical to immunofluorescence results are sample preparation and specific antibodies. Antibody staining of cells requires fixation and permeabilization, both of which can result in loss or redistribution of proteins and masking of epitopes. A high-throughput approach demands a standardized protocol suitable for the majority of proteins across cellular compartments. Paper I presents an evaluation of sample preparation techniques from which such a single fixation and permeabilization protocol was optimized. Paper II describes the results from applying this protocol to 4000 human proteins in three cell lines of different origin. Paper III presents a strategy for application-specific antibody validation. Antibodies are the key reagents in immunofluorescence, but all antibodies have potential for off-target binding and should be validated thoroughly. Antibody performance varies across sample types and applications due to the competition present and the effect of the sample preparation on antigen accessibility. In this paper application-specific validation for immunofluorescence was conducted using colocalization with fluorescently tagged protein in transgenic cell lines. QC 20160509

Published
2016

8. Antibody Validation in Bioimaging Applications Based on Endogenous Expression of Tagged Proteins

Author

Skogs, Marie, Stadler, Charlotte, Schutten, Rutger, Hjelmare, Martin, Gnann, Christian, Björk, Lars, Poser, Ina, Hyman, Anthony, Uhlén, Mathias, Lundberg, Emma, Skogs, Marie, Stadler, Charlotte, Schutten, Rutger, Hjelmare, Martin, Gnann, Christian, Björk, Lars, Poser, Ina, Hyman, Anthony, Uhlén, Mathias, and Lundberg, Emma

Abstract

Antibodies are indispensible research tools, yet the scientific community has not adopted standardized procedures to validate their specificity. Here we present a strategy to systematically validate antibodies for immunofluorescence (IF) applications using gene tagging. We have assessed the on- and off-target binding capabilities of 197 antibodies using 108 cell lines expressing EGFP-tagged target proteins at endogenous levels. Furthermore, we assessed batch-to-batch effects for 35 target proteins, showing that both the on- and off-target binding patterns vary significantly between antibody batches and that the proposed strategy serves as a reliable procedure for ensuring reproducibility upon production of new antibody batches. In summary, we present a systematic scheme for antibody validation in IF applications using endogenous expression of tagged proteins. This is an important step toward a reproducible approach for context- and application-specific antibody validation and improved reliability of antibody-based experiments and research data., QC 20170216

Published
2017
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9. The Cell Atlas : Creation of an image-based atlas of the subcellular distribution of the human proteome.

Author

Lundberg, Emma, Ait Blal, Carl, Alm, Tove L., Björk, Lars, Bäckström, Anna, Danielsson, Frida, Fall, Jenny, Hjelmare, Martin, Mahdessian, Diana, Schutten, Rutger, Skogs, Marie, Stadler, Charlotte, Sullivan, Devin P., Thul, Peter, Wiking, Mikaela, Winsnes, Casper F., Åkesson, Lovisa, Uhlén, Mathias, Lundberg, Emma, Ait Blal, Carl, Alm, Tove L., Björk, Lars, Bäckström, Anna, Danielsson, Frida, Fall, Jenny, Hjelmare, Martin, Mahdessian, Diana, Schutten, Rutger, Skogs, Marie, Stadler, Charlotte, Sullivan, Devin P., Thul, Peter, Wiking, Mikaela, Winsnes, Casper F., Åkesson, Lovisa, and Uhlén, Mathias

Abstract

QC 20200312

Published
2016

10. An image-based view of the microtubule proteome

Author

Danielsson, Frida, Skogs, Marie, Åkesson, Lovisa, Mahdessian, Diana, Sullivan, Devin P., Thul, Peter, Wiking, Mikaela, Björk, Lars, Schutten, Rutger, Ait Blal, Carl, Hjelmare, Martin, Gnann, Christian, Uhlén, Mathias, Lundberg, Emma, Danielsson, Frida, Skogs, Marie, Åkesson, Lovisa, Mahdessian, Diana, Sullivan, Devin P., Thul, Peter, Wiking, Mikaela, Björk, Lars, Schutten, Rutger, Ait Blal, Carl, Hjelmare, Martin, Gnann, Christian, Uhlén, Mathias, and Lundberg, Emma

Abstract

QC 20170523

Published
2016

11. Application specific antibody validation. The Human Protein Atlas validation scheme and how to confirm subcellular protein localization.

Author

Bjork, L., Ait Blal, C., Alm, Tove L., Bäckström, Anna, Gnann, C., Hjelmare, Martin, Schutten, Rutger, Skogs, Marie, Stadler, Charlotte, Uhlén, Mathias, Lundberg, Emma, Bjork, L., Ait Blal, C., Alm, Tove L., Bäckström, Anna, Gnann, C., Hjelmare, Martin, Schutten, Rutger, Skogs, Marie, Stadler, Charlotte, Uhlén, Mathias, and Lundberg, Emma

Abstract

QC 20170410

Published
2016

12. Profiling the human cytoplasmic proteome.

Author

Mahdessian, Diana, Wiking, Mikaela, Åkesson, Lina, Danielsson, Frida, Ait Blal, Carl, Sullivan, Devin P., Thul, Peter, Gnann, Christian, Bäckström, Anna, Fall, Jenny, Schutten, Rutger, Björk, Lars, Hjelmare, Martin, Skogs, Marie, Stadler, Charlotte, Uhlén, Mathias, Lundberg, Emma, Mahdessian, Diana, Wiking, Mikaela, Åkesson, Lina, Danielsson, Frida, Ait Blal, Carl, Sullivan, Devin P., Thul, Peter, Gnann, Christian, Bäckström, Anna, Fall, Jenny, Schutten, Rutger, Björk, Lars, Hjelmare, Martin, Skogs, Marie, Stadler, Charlotte, Uhlén, Mathias, and Lundberg, Emma

Abstract

QC 20170410

Published
2016

13. Drafting the intermediate filament proteome.

Author

Wiking, Mikaela, Ait Blal, Carl, Björk, Lars, Bäckström, Anna, Danielsson, Frida, Fall, Jenny, Gnann, Christian, Hjelmare, Martin, Mahdessian, Diana, Schutten, Rutger, Skogs, Marie, Stadler, Charlotte, Sullivan, D. P., Thul, Peter, Winsnes, Casper F., Åkesson, Lina, Uhlén, Mathias, Lundberg, Emma, Wiking, Mikaela, Ait Blal, Carl, Björk, Lars, Bäckström, Anna, Danielsson, Frida, Fall, Jenny, Gnann, Christian, Hjelmare, Martin, Mahdessian, Diana, Schutten, Rutger, Skogs, Marie, Stadler, Charlotte, Sullivan, D. P., Thul, Peter, Winsnes, Casper F., Åkesson, Lina, Uhlén, Mathias, and Lundberg, Emma

Abstract

QC 20211020

Published
2016

17. The Subcellular Protein Atlas

Author

Wiking, Mikaela, Skogs, Marie, Hjelmare, Martin, Blal, Hammou Ait, Stadler, Charlotte, Mahdessian, Diana, Danielsson, Frida, Schutten, Rutger, Åbergh, Annica, Uhlén, Mathias, Lundberg, Emma, Wiking, Mikaela, Skogs, Marie, Hjelmare, Martin, Blal, Hammou Ait, Stadler, Charlotte, Mahdessian, Diana, Danielsson, Frida, Schutten, Rutger, Åbergh, Annica, Uhlén, Mathias, and Lundberg, Emma

Abstract

QC 20150512

Published
2014

18. Contribution of Antibody-based Protein Profiling to the Human Chromosome-centric Proteome Project (C-HPP)

Author

Fagerberg, Linn, Oksvold, Per, Skogs, Marie, Algenäs, Cajsa, Lundberg, Emma, Pontén, Fredrik, Sivertsson, Asa, Odeberg, Jacob, Klevebring, Daniel, Kampf, Caroline, Asplund, Anna, Sjöstedt, Evelina, Al-Khalili Szigyarto, Cristina, Edqvist, Per-Henrik, Olsson, IngMarie, Rydberg, Urban, Hudson, Paul, Ottosson Takanen, Jenny, Berling, Holger, Björling, Lisa, Tegel, Hanna, Rockberg, Johan, Nilsson, Peter, Navani, Sanjay, Jirström, Karin, Mulder, Jan, Schwenk, Jochen M, Zwahlen, Martin, Hober, Sophia, Forsberg, Mattias, von Feilitzen, Kalle, Uhlén, Mathias, Fagerberg, Linn, Oksvold, Per, Skogs, Marie, Algenäs, Cajsa, Lundberg, Emma, Pontén, Fredrik, Sivertsson, Asa, Odeberg, Jacob, Klevebring, Daniel, Kampf, Caroline, Asplund, Anna, Sjöstedt, Evelina, Al-Khalili Szigyarto, Cristina, Edqvist, Per-Henrik, Olsson, IngMarie, Rydberg, Urban, Hudson, Paul, Ottosson Takanen, Jenny, Berling, Holger, Björling, Lisa, Tegel, Hanna, Rockberg, Johan, Nilsson, Peter, Navani, Sanjay, Jirström, Karin, Mulder, Jan, Schwenk, Jochen M, Zwahlen, Martin, Hober, Sophia, Forsberg, Mattias, von Feilitzen, Kalle, and Uhlén, Mathias

Abstract

A gene-centric Human Proteome Project has been proposed to characterize the human protein-coding genes in a chromosome-centered manner to understand human biology and disease. Here, we report on the protein evidence for all genes predicted from the genome sequence based on manual annotation from literature (UniProt), antibody-based profiling in cells, tissues and organs and analysis of the transcript profiles using next generation sequencing in human cell lines of different origins. We estimate that there is good evidence for protein existence for 69% (n = 13985) of the human protein-coding genes, while 23% have only evidence on the RNA level and 7% still lack experimental evidence. Analysis of the expression patterns shows few tissue-specific proteins and approximately half of the genes expressed in all the analyzed cells. The status for each gene with regards to protein evidence is visualized in a chromosome-centric manner as part of a new version of the Human Protein Atlas ( www.proteinatlas.org ).

Published
2013
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19. RNA Deep Sequencing as a Tool for Selection of Cell Lines for Systematic Subcellular Localization of All Human Proteins

Author

Danielsson, Frida, Wiking, Mikaela, Mahdessian, Diana, Skogs, Marie, Ait Blal, Hammou, Hjelmare, Martin, Stadler, Charlotte, Uhlén, Mathias, Lundberg, Emma, Danielsson, Frida, Wiking, Mikaela, Mahdessian, Diana, Skogs, Marie, Ait Blal, Hammou, Hjelmare, Martin, Stadler, Charlotte, Uhlén, Mathias, and Lundberg, Emma

Abstract

One of the major challenges of a chromosome-centric proteome project is to explore in a systematic manner the potential proteins identified from the chromosomal genome sequence, but not yet characterized on a protein level. Here, we describe the use of RNA deep sequencing to screen human cell lines for RNA profiles and to use this information to select cell lines suitable for characterization of the corresponding gene product. In this manner, the subcellular localization of proteins can be analyzed systematically using antibody-based confocal microscopy. We demonstrate the usefulness of selecting cell lines with high expression levels of RNA transcripts to increase the likelihood of high quality immunofluorescence staining and subsequent successful subcellular localization of the corresponding protein. The results show a path to combine transcriptomics with affinity proteomics to characterize the proteins in a gene- or chromosome-centric manner., QC 20130305

Published
2013
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20. Mapping the subcellular protein distribution in three human cell lines

Author

Fagerberg, Linn, Sandler, Charlotte, Skogs, Marie, Hjelmare, Martin, Jonasson, Kalle, Wiking, Mikaela, Åbergh, Annica, Uhlén, Mathias, Lundberg, Emma, Fagerberg, Linn, Sandler, Charlotte, Skogs, Marie, Hjelmare, Martin, Jonasson, Kalle, Wiking, Mikaela, Åbergh, Annica, Uhlén, Mathias, and Lundberg, Emma

Abstract

The subcellular locations of proteins are closely related to their function and constitute an essential aspect for understanding the complex machinery of living cells. A systematic effort has been initiated to map the protein distribution in three functionally different cell lines with the aim to provide a subcellular localization index for at least one representative protein from all human protein-encoding genes. Here, we present the results of over 4,000 proteins mapped to 16 subcellular compartments. The results indicate a ubiquitous protein expression with a majority of the proteins found in all three cell lines and a large portion localized to two or more compartments. The inter-relationships between the subcellular compartments are visualized in a protein-compartment network based on all detected proteins. Hierarchical clustering was performed to determine how closely related the organelles are in terms of protein constituents and compare the proteins detected in each cell type. Our results show distinct organelle proteomes, well conserved across the cell types, and demonstrate that biochemically similar organelles are grouped together., QC 20110905

Published
2011
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21. Contribution of Antibody-based Protein Profiling to the Human Chromosome-centric Proteome Project (C-HPP)

Author

Fagerberg, Linn, primary, Oksvold, Per, additional, Skogs, Marie, additional, Älgenäs, Cajsa, additional, Lundberg, Emma, additional, Pontén, Fredrik, additional, Sivertsson, Åsa, additional, Odeberg, Jacob, additional, Klevebring, Daniel, additional, Kampf, Caroline, additional, Asplund, Anna, additional, Sjöstedt, Evelina, additional, Al-Khalili Szigyarto, Cristina, additional, Edqvist, Per-Henrik, additional, Olsson, IngMarie, additional, Rydberg, Urban, additional, Hudson, Paul, additional, Ottosson Takanen, Jenny, additional, Berling, Holger, additional, Björling, Lisa, additional, Tegel, Hanna, additional, Rockberg, Johan, additional, Nilsson, Peter, additional, Navani, Sanjay, additional, Jirström, Karin, additional, Mulder, Jan, additional, Schwenk, Jochen M., additional, Zwahlen, Martin, additional, Hober, Sophia, additional, Forsberg, Mattias, additional, von Feilitzen, Kalle, additional, and Uhlén, Mathias, additional

Published
2012
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25. An antibody validation scheme for immunofluorescence using gene tagging

Author

Skogs, Marie, Stadler, Charlotte, Schutten, Rutger, Hjelmare, Martin, Gnann, Christian, Poser, Ina, Hyman, Anthony, Uhlén, Mathias, Lundberg, Emma, Skogs, Marie, Stadler, Charlotte, Schutten, Rutger, Hjelmare, Martin, Gnann, Christian, Poser, Ina, Hyman, Anthony, Uhlén, Mathias, and Lundberg, Emma

Abstract

QC 20160518

27. Antibody Validation in Bioimaging Applications Based on Endogenous Expression of Tagged Proteins.

Author

Skogs M, Stadler C, Schutten R, Hjelmare M, Gnann C, Björk L, Poser I, Hyman A, Uhlén M, and Lundberg E

Subjects
Analysis of Variance, Antibodies chemistry, Atlases as Topic, Cell Line, Gene Expression, Green Fluorescent Proteins genetics, Green Fluorescent Proteins metabolism, Humans, Reference Standards, Reproducibility of Results, Antibodies analysis, Fluorescent Antibody Technique standards, Microscopy, Confocal standards, Staining and Labeling methods
Abstract

Antibodies are indispensible research tools, yet the scientific community has not adopted standardized procedures to validate their specificity. Here we present a strategy to systematically validate antibodies for immunofluorescence (IF) applications using gene tagging. We have assessed the on- and off-target binding capabilities of 197 antibodies using 108 cell lines expressing EGFP-tagged target proteins at endogenous levels. Furthermore, we assessed batch-to-batch effects for 35 target proteins, showing that both the on- and off-target binding patterns vary significantly between antibody batches and that the proposed strategy serves as a reliable procedure for ensuring reproducibility upon production of new antibody batches. In summary, we present a systematic scheme for antibody validation in IF applications using endogenous expression of tagged proteins. This is an important step toward a reproducible approach for context- and application-specific antibody validation and improved reliability of antibody-based experiments and research data.

Published
2017
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28. Contribution of antibody-based protein profiling to the human Chromosome-centric Proteome Project (C-HPP).

Author

Fagerberg L, Oksvold P, Skogs M, Algenäs C, Lundberg E, Pontén F, Sivertsson A, Odeberg J, Klevebring D, Kampf C, Asplund A, Sjöstedt E, Al-Khalili Szigyarto C, Edqvist PH, Olsson I, Rydberg U, Hudson P, Ottosson Takanen J, Berling H, Björling L, Tegel H, Rockberg J, Nilsson P, Navani S, Jirström K, Mulder J, Schwenk JM, Zwahlen M, Hober S, Forsberg M, von Feilitzen K, and Uhlén M

Subjects
Cell Line, Cell Line, Tumor, Gene Expression, Gene Expression Profiling, Genome, Human, Humans, Microscopy, Fluorescence, Neoplasm Proteins genetics, Neoplasm Proteins metabolism, Neoplasms genetics, Neoplasms metabolism, Oligonucleotide Array Sequence Analysis, Proteome genetics, Proteome metabolism, RNA, Messenger genetics, RNA, Messenger metabolism, Antibodies chemistry, Chromosomes, Human chemistry, Human Genome Project, Neoplasm Proteins isolation & purification, Neoplasms chemistry, Proteome isolation & purification
Abstract

A gene-centric Human Proteome Project has been proposed to characterize the human protein-coding genes in a chromosome-centered manner to understand human biology and disease. Here, we report on the protein evidence for all genes predicted from the genome sequence based on manual annotation from literature (UniProt), antibody-based profiling in cells, tissues and organs and analysis of the transcript profiles using next generation sequencing in human cell lines of different origins. We estimate that there is good evidence for protein existence for 69% (n = 13985) of the human protein-coding genes, while 23% have only evidence on the RNA level and 7% still lack experimental evidence. Analysis of the expression patterns shows few tissue-specific proteins and approximately half of the genes expressed in all the analyzed cells. The status for each gene with regards to protein evidence is visualized in a chromosome-centric manner as part of a new version of the Human Protein Atlas ( www.proteinatlas.org ).

Published
2013
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29. RNA deep sequencing as a tool for selection of cell lines for systematic subcellular localization of all human proteins.

Author

Danielsson F, Wiking M, Mahdessian D, Skogs M, Ait Blal H, Hjelmare M, Stadler C, Uhlén M, and Lundberg E

Subjects
Base Sequence, Cell Line metabolism, Chromosomes, Human, Genome, Human, High-Throughput Nucleotide Sequencing, Humans, Sequence Analysis, RNA, Gene Expression Profiling, Proteins genetics, Proteins metabolism, Proteome, RNA genetics, RNA metabolism
Abstract

One of the major challenges of a chromosome-centric proteome project is to explore in a systematic manner the potential proteins identified from the chromosomal genome sequence, but not yet characterized on a protein level. Here, we describe the use of RNA deep sequencing to screen human cell lines for RNA profiles and to use this information to select cell lines suitable for characterization of the corresponding gene product. In this manner, the subcellular localization of proteins can be analyzed systematically using antibody-based confocal microscopy. We demonstrate the usefulness of selecting cell lines with high expression levels of RNA transcripts to increase the likelihood of high quality immunofluorescence staining and subsequent successful subcellular localization of the corresponding protein. The results show a path to combine transcriptomics with affinity proteomics to characterize the proteins in a gene- or chromosome-centric manner.

Published
2013
Full Text
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