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Towards subcellular localization of the human proteome using bioimaging
KTH, School of Biotechnology (BIO), Proteomics (closed 20130101). KTH, Centres, Science for Life Laboratory, SciLifeLab. (Cell Profiing)
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Since the publication of the complete sequence of the human genome in 2003 there has been great interest in exploring the functions of the proteins encoded by the genes. To reveal the function of each and every protein, investigation of protein localization at the subcellular level has become a central focus in this research area, since the localization and function of a protein is closely related. The objective of the studies presented in this doctoral thesis was to systematically explore the human proteome at the subcellular level using bioimaging and to develop techniques for validation of the results obtained.

A common imaging technique for protein detection is immunofluorescence (IF), where antibodies are used to target proteins in fixated cells. A fixation protocol suitable for large-scale IF studies was developed and optimized to work for a broad set of proteins. As the technique relies on antibodies, validation of their specificity to the target protein is crucial. A platform based on siRNA gene silencing in combination with IF was set-up to evaluate antibody specificity by quantitative image analysis before and after suppression of its target protein. As a proof of concept, the platform was then used for validation of 75 antibodies, proving it to be applicable for validation of antibodies in a systematic manner.

Because of the fixation, there is a common concern about how well IF data reflects the in vivo subcellular distribution of proteins. To address this, 500 proteins were tagged with green fluorescent protein (GFP) and used to compare protein localization results between IF to those achieved using GFP tagged proteins in live cells. It was concluded that protein localization data from fixated cells satisfactory represented the situation in vivo and together exhibit a powerful approach for confirming localizations of yet uncharacterized proteins.

Finally, a global analysis based on IF data of approximately 20 % of the human proteome was performed, providing a first overview of the subcellular landscape in three different cell lines. It was found that the intracellular distribution of proteins is complex, with many proteins occurring in several organelles. The results also confirmed the close relationship between protein function and localization, which in a way further strengthens the accuracy of the IF approach for detection of proteins at the subcellular level.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2012. , 60 p.
Series
Trita-BIO-Report, ISSN 1654-2312 ; 12:19
Keyword [en]
Antibody, antibody validation, automated image analysis, automated microscopy, cell line, confocal microscopy, fixation, green fluorescent protein (GFP), immunofluorescence (IF), organelle, protein expression, siRNA, subcellular localization
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:kth:diva-103616ISBN: 978-91-7501-483-8 (print)OAI: oai:DiVA.org:kth-103616DiVA: diva2:560974
Public defence
2012-11-09, MTC, Karolinska institutet, Stockholm, 10:00 (English)
Opponent
Supervisors
Projects
The Human Protein Atlas
Funder
Science for Life Laboratory - a national resource center for high-throughput molecular bioscience
Note

QC 20121017

Available from: 2012-10-17 Created: 2012-10-16 Last updated: 2013-04-15Bibliographically approved
List of papers
1. A single fixation protocol for proteome-wide immunofluorescence localization studies
Open this publication in new window or tab >>A single fixation protocol for proteome-wide immunofluorescence localization studies
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2010 (English)In: Journal of Proteomics, ISSN 1874-3919, Vol. 73, no 6, 1067-1078 p.Article in journal (Refereed) Published
Abstract [en]

Immunofluorescence microscopy is a valuable tool for analyzing protein expression and localization at a subcellular level thus providing information regarding protein function, interaction partners and its role in cellular processes. When performing sample fixation, parameters such as difference in accessibility of proteins present in various cellular compartments as well as the chemical composition of the protein to be studied, needs to be taken into account. However, in systematic and proteome-wide efforts, a need exists for standard fixation protocol(s) that works well for the majority of all proteins independent of subcellular localization. Here, we report on a study with the goal to find a standardized protocol based on the analysis of 18 human proteins localized in 11 different organelles and subcellular structures. Six fixation protocols were tested based on either dehydration by alcohols (methanol, ethanol or iso-propanol) or cross-linking by paraformaldehyde followed by detergent permeabilization (Triton X-100 or saponin) in three human cell lines. Our results show that cross-linking is essential for proteome-wide localization studies and that cross-linking using paraformaldehyde followed by Triton X-100 permeabilization successfully can be used as a single fixation protocol for systematic studies.

Keyword
Antibody, Confocal microscopy, Fixation, Immunofluorescence, Organelle, Permeabilization
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-29841 (URN)10.1016/j.jprot.2009.10.012 (DOI)000277763800004 ()2-s2.0-77950516661 (Scopus ID)
Note
QC 20110223 3rd EuPA Congress, Stockholm, SWEDEN, JUN 14-17, 2009Available from: 2011-02-23 Created: 2011-02-17 Last updated: 2016-05-16Bibliographically approved
2. Systematic validation of antibody binding and protein subcellular localization using siRNA and confocal microscopy
Open this publication in new window or tab >>Systematic validation of antibody binding and protein subcellular localization using siRNA and confocal microscopy
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2012 (English)In: Journal of Proteomics, ISSN 1874-3919, E-ISSN 1876-7737, Vol. 75, no 7, 2236-2251 p.Article in journal (Refereed) Published
Abstract [en]

We have developed a platform for validation of antibody binding and protein subcellular localization data obtained from immunofluorescence using siRNA technology combined with automated confocal microscopy and image analysis. By combining the siRNA technology with automated sample preparation, automated imaging and quantitative image analysis, a high-throughput assay has been set-up to enable confirmation of accurate protein binding and localization in a systematic manner. Here, we describe the analysis and validation of the subcellular location of 65 human proteins, targeted by 75 antibodies and silenced by 130 siRNAs. A large fraction of (80%) the subcellular locations, including locations of several previously uncharacterized proteins, could be confirmed by the significant down-regulation of the antibody signal after the siRNA silencing. A quantitative analysis was set-up using automated image analysis to facilitate studies of targets found in more than one compartment. The results obtained using the platform demonstrate that siRNA silencing in combination with quantitative image analysis of antibody signals in different compartments of the cells is an attractive approach for ensuring accurate protein localization as well as antibody binding using immunofluorescence. With a large fraction of the human proteome still unexplored, we suggest this approach to be of great importance under the continued work of mapping the human proteome on a subcellular level.

Keyword
siRNA, Immunofluorescence, Subcellular location, Quantitative image analysis, Antibody validation
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-95259 (URN)10.1016/j.jprot.2012.01.030 (DOI)000303136600021 ()22361696 (PubMedID)2-s2.0-84858748188 (Scopus ID)
Funder
Knut and Alice Wallenberg FoundationEU, European Research CouncilScience for Life Laboratory - a national resource center for high-throughput molecular bioscience
Note

QC 20120521

Available from: 2012-05-21 Created: 2012-05-21 Last updated: 2017-12-07Bibliographically approved
3. Immunofluorescence and fluorescent protein-tagging are complementary techniques with high correlation for subcellular investigation of the human proteome in mammalian cells
Open this publication in new window or tab >>Immunofluorescence and fluorescent protein-tagging are complementary techniques with high correlation for subcellular investigation of the human proteome in mammalian cells
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(English)Article in journal (Other academic) Submitted
Abstract [en]

Imaging techniques such as immunofluorescence (IF) and expression of fluorescent protein (FP) fusions are widely used to investigate the subcellular distribution of proteins. Due to potential steric hindrance with FPs and fixation artifacts obtained during IF, the reliability of these two fundamental methods is often discussed. Here we report a systematic study of over 500 human proteins where the localizations obtained in live versus fixed cells using FPs and IF respectively have been compared. The results indicate that 80% of the analyzed proteins yield the same subcellular distribution, which is as high as that seen for FP tagging at either the N- and C-terminal. The localizations of 250 proteins, with no previous experimental data, were determined by the overlap of the two methods as applied here. The fraction of proteins located to multiple organelles is approximately 60% for both methods, indicating a complex subcellular protein organization. The result shows that IF and FP tagging are reliable techniques, both needed for a complete investigation of the subcellular human proteome.

National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-103628 (URN)
Note

QS 2012

Available from: 2012-10-17 Created: 2012-10-17 Last updated: 2012-10-17Bibliographically approved
4. Mapping the subcellular protein distribution in three human cell lines
Open this publication in new window or tab >>Mapping the subcellular protein distribution in three human cell lines
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2011 (English)In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 10, no 8, 3766-3777 p.Article in journal (Refereed) Published
Abstract [en]

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.

Keyword
antibody, organelle, Human Protein Atlas, subcellular atlas, immunofluorescence, confocal microscopy
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-31514 (URN)10.1021/pr200379a (DOI)000293487900041 ()2-s2.0-79961240625 (Scopus ID)
Funder
Knut and Alice Wallenberg FoundationScience for Life Laboratory - a national resource center for high-throughput molecular bioscience
Note

QC 20110905

Available from: 2011-03-17 Created: 2011-03-17 Last updated: 2017-12-11Bibliographically approved

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