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Mapping the subcellular protein distribution in three human cell lines
KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101).ORCID-id: 0000-0003-0198-7137
KTH, Centra, Science for Life Laboratory, SciLifeLab.
KTH, Centra, Science for Life Laboratory, SciLifeLab.ORCID-id: 0000-0002-2998-3077
KTH, Centra, Science for Life Laboratory, SciLifeLab.ORCID-id: 0000-0001-7375-9681
Visa övriga samt affilieringar
2011 (Engelska)Ingår i: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 10, nr 8, s. 3766-3777Artikel i tidskrift (Refereegranskat) 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.

Ort, förlag, år, upplaga, sidor
2011. Vol. 10, nr 8, s. 3766-3777
Nyckelord [en]
antibody, organelle, Human Protein Atlas, subcellular atlas, immunofluorescence, confocal microscopy
Nationell ämneskategori
Industriell bioteknik
Identifikatorer
URN: urn:nbn:se:kth:diva-31514DOI: 10.1021/pr200379aISI: 000293487900041Scopus ID: 2-s2.0-79961240625OAI: oai:DiVA.org:kth-31514DiVA, id: diva2:404466
Forskningsfinansiär
Knut och Alice Wallenbergs StiftelseScience for Life Laboratory - a national resource center for high-throughput molecular bioscience
Anmärkning

QC 20110905

Tillgänglig från: 2011-03-17 Skapad: 2011-03-17 Senast uppdaterad: 2020-01-10Bibliografiskt granskad
Ingår i avhandling
1. Mapping the human proteome using bioinformatic methods
Öppna denna publikation i ny flik eller fönster >>Mapping the human proteome using bioinformatic methods
2011 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
Abstract [en]

The fundamental goal of proteomics is to gain an understanding of the expression and function of the proteome on the level of individual proteins, on the level of defined cell types and on the level of the entire organism. In this thesis, the human proteome is explored using membrane protein topology prediction methods to define the human membrane proteome and by global protein expression profiling, which relies on a complex study of the location and expression levels of proteins in tissues and cells.

A whole-proteome analysis was performed based on the predicted protein-coding genes of humans using a selection of membrane protein topology prediction methods. The study used a majority decision-based method, which estimated that approximately 26% of the human genes encode for a membrane protein. The prediction results are displayed in a visualization tool to facilitate the selection of antigens to be used for antibody generation.

Global protein expression profiles in a large number of cells and tissues in the human body were analyzed for more than 4000 protein targets, based on data from the antibody-based immunohistochemistry and immunofluorescence methods within the framework of the Human Protein Atlas project. The results revealed few cell-type specific proteins and a high fraction of human proteins expressed in most cells, suggesting that cell and tissue specificity is attained by a fine-tuned regulation of protein levels. The expression profiles were also used to analyze the relationship between 45 cell lines by hierarchical clustering and principal component analysis. The global protein expression patterns overall reflected the tumor origin of the cells, and also allowed for identification of proteins of importance for distinguishing different categories of cell lines, as defined by phenotype of progenitor cell. In addition, the protein distribution in 16 subcellular compartments in three of the human cell lines was mapped. A large fraction of proteins were localized in two or more compartments and, in line with previous results, a majority of proteins were detected in all three cell lines.

Finally, mass spectrometry-based protein expression levels were compared to RNA-seq-based transcript expression levels in three cell lines. Highly ubiquitous mRNA expression was found and the changes of expression levels between the cell lines showed high correlations between proteins and transcripts. Large general differences in abundance of proteins from various functional classes were observed. A comparison between categories based on expression levels revealed that, in general, genes with varying expression levels between the cell lines or only expressed in one cell line were highly enriched for cell-surface proteins.

These studies show a path for a systematic analysis to characterize the proteome in human cells, tissues and organs.

Ort, förlag, år, upplaga, sidor
Stockholm: KTH Royal Institute of Technology., 2011. s. 66
Serie
Trita-BIO-Report, ISSN 1654-2312 ; 2011:4
Nyckelord
proteome, transcriptome, bioinformatics, membrane protein prediction, subcellular localization, protein expression level, cell line, immunohistochemistry, immunofluorescence
Nationell ämneskategori
Bioinformatik och systembiologi
Forskningsämne
SRA - Molekylär biovetenskap
Identifikatorer
urn:nbn:se:kth:diva-31477 (URN)978-91-7415-886-1 (ISBN)
Disputation
2011-04-08, F3, Lindstedtsvägen 26, KTH, Stockholm, 14:41 (Engelska)
Opponent
Handledare
Projekt
The Human Protein Atlas project
Forskningsfinansiär
Knut och Alice Wallenbergs Stiftelse
Anmärkning
QC 20110317Tillgänglig från: 2011-03-17 Skapad: 2011-03-16 Senast uppdaterad: 2011-03-17Bibliografiskt granskad
2. Towards subcellular localization of the human proteome using bioimaging
Öppna denna publikation i ny flik eller fönster >>Towards subcellular localization of the human proteome using bioimaging
2012 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
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.

Ort, förlag, år, upplaga, sidor
Stockholm: KTH Royal Institute of Technology, 2012. s. 60
Serie
Trita-BIO-Report, ISSN 1654-2312 ; 12:19
Nyckelord
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
Nationell ämneskategori
Biokemi och molekylärbiologi
Identifikatorer
urn:nbn:se:kth:diva-103616 (URN)978-91-7501-483-8 (ISBN)
Disputation
2012-11-09, MTC, Karolinska institutet, Stockholm, 10:00 (Engelska)
Opponent
Handledare
Projekt
The Human Protein Atlas
Forskningsfinansiär
Science for Life Laboratory - a national resource center for high-throughput molecular bioscience
Anmärkning

QC 20121017

Tillgänglig från: 2012-10-17 Skapad: 2012-10-16 Senast uppdaterad: 2013-04-15Bibliografiskt granskad
3. Antibody-based subcellular localization of the human proteome
Öppna denna publikation i ny flik eller fönster >>Antibody-based subcellular localization of the human proteome
2016 (Engelska)Licentiatavhandling, sammanläggning (Övrigt vetenskapligt)
Abstract [en]

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. 

Ort, förlag, år, upplaga, sidor
Stockholm: KTH Royal Institute of Technology, 2016. s. viii, 53
Serie
TRITA-BIO-Report, ISSN 1654-2312 ; 2016:13
Nyckelord
Human proteome, Subcellular localization, Organelles, Immunofluorescence, Fixation, Permeabilization, Antibody validation
Nationell ämneskategori
Cellbiologi
Forskningsämne
Bioteknologi
Identifikatorer
urn:nbn:se:kth:diva-186138 (URN)978-91-7729-010-0 (ISBN)
Presentation
2016-06-08, Alfa2, Tomtebodavägen 23A, Solna, 14:00 (Engelska)
Opponent
Handledare
Forskningsfinansiär
Knut och Alice Wallenbergs Stiftelse
Anmärkning

QC 20160509

Tillgänglig från: 2016-05-16 Skapad: 2016-05-02 Senast uppdaterad: 2016-05-16Bibliografiskt granskad

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Fagerberg, LinnSandler, CharlotteSkogs, MarieHjelmare, MartinJonasson, KalleWiking, MikaelaÅbergh, AnnicaUhlén, MathiasLundberg, Emma
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Proteomik (stängd 20130101)Science for Life Laboratory, SciLifeLab
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