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Spatial proteome profiling of the compartments of the human cell using an antibody-based approach
KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. (Emma Lundberg)ORCID iD: 0000-0002-6368-6690
2017 (English)Licentiate thesis, comprehensive summary (Other academic)
Abstract [en]

The human cell is complex, with countless processes ongoing in parallel in specialized compartments, the organelles. Cells can be studied in vitro by using immortalized cell lines that represent cells in vivo to a varying degree. Gene expression varies between cell types and an average cell line expresses around 10,000-12,000 genes, as measured with RNA sequencing. These genes encode the cell’s proteome; the full set of proteins that perform functions in the cell. In paper I we show that RNA sequencing is a necessary tool for studying the proteome of the human cell.

By studying the proteome, and proteins’ localization in the cell, information can be assembled on how the cell functions. Image-based methods allow for detailed spatial resolution of protein localization as well as enable the study of temporal events. Visualization of a protein can be accomplished by using either a cell line that is transfected to express the protein with a fluorescent tag, or by targeting the protein with an affinity reagent such as an antibody. In paper II we present subcellular data for a majority of the human proteins, showing that there is a high degree of complexity in regard to where proteins localize in the cell.

Cellular energy is generated in the mitochondria, an important organelle that is also active in many other different functions. Today approximately only a third of the estimated mitochondrial proteome has been validated experimentally, indicating that there is much more to understand with regard to the functions of the mitochondria. In paper III we explore the mitochondrial proteome, based on the results of paper II. We also present a method for sublocalizing proteins to subcompartments that can be performed in a high-throughput manner.

To conclude, this thesis shows that transcriptomics is a useful tool for proteome-wide subcellular localization, and presents high-resolution spatial distribution data for the human cell with a deeper analysis of the mitochondrial proteome.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2017. , 49 p.
Series
TRITA-BIO-Report, ISSN 1654-2312 ; 2017:11
Keyword [en]
Fluorescence imaging, Human proteome, Human Protein Atlas, Immunofluorescence, Mitochondria, Organelles, Spatial distribution
National Category
Cell Biology
Research subject
Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-206817ISBN: 978-91-7729-393-4 (print)OAI: oai:DiVA.org:kth-206817DiVA: diva2:1095136
Presentation
2017-06-16, Gamma 2, Tomtebodavägen 23A, Solna, 10:00 (English)
Opponent
Supervisors
Funder
Knut and Alice Wallenberg Foundation
Note

QC 20170512

Available from: 2017-05-12 Created: 2017-05-12 Last updated: 2017-05-12Bibliographically approved
List of papers
1. RNA Deep Sequencing as a Tool for Selection of Cell Lines for Systematic Subcellular Localization of All Human Proteins
Open this publication in new window or tab >>RNA Deep Sequencing as a Tool for Selection of Cell Lines for Systematic Subcellular Localization of All Human Proteins
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2013 (English)In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 12, no 1, 231-239 p.Article in journal (Refereed) Published
Abstract [en]

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.

Keyword
antibody, Human Protein Atlas, Human Proteome Project, RNA sequencing, subcellular localization
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-119069 (URN)10.1021/pr3009308 (DOI)000313156300026 ()2-s2.0-84874045044 (ScopusID)
Funder
Knut and Alice Wallenberg FoundationScience for Life Laboratory - a national resource center for high-throughput molecular bioscience
Note

QC 20130305

Available from: 2013-03-05 Created: 2013-03-05 Last updated: 2017-05-12Bibliographically approved
2. A subcellular map of the human proteome
Open this publication in new window or tab >>A subcellular map of the human proteome
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(English)Manuscript (preprint) (Other academic)
National Category
Cell Biology
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-207011 (URN)10.1126/science.aal3321 (DOI)
Note

QC 20170512

Available from: 2017-05-12 Created: 2017-05-12 Last updated: 2017-05-12Bibliographically approved
3. An image-based map of the human mitochondrial proteome
Open this publication in new window or tab >>An image-based map of the human mitochondrial proteome
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Defining the human mitochondrial proteome is crucial to understand the mitochondria’s diverse functions and role in disease. Here, we present an image-based map of the human mitochondrial proteome containing 1,070 proteins, of which 376 have previously not been reported to localize to the mitochondria. Our analysis shows that approximately 50% (n=493) of the proteins localize to additional cellular compartments, likely contributing to the diverse cellular functions of mitochondria. This map of the mitochondrial proteome, part of the Cell Atlas of the Human Protein Atlas database (www.proteinatlas.org), provides a valuable knowledge resource for studies of mitochondria function, dysfunction and disease.

Keyword
Mitochondria, Human Protein Atlas, Human Proteome, Immunofluorescence, Multilocalizing protein
National Category
Cell Biology
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-206811 (URN)
Funder
Knut and Alice Wallenberg Foundation
Note

QC 20170512

Available from: 2017-05-08 Created: 2017-05-08 Last updated: 2017-05-12Bibliographically approved

Open Access in DiVA

The full text will be freely available from 2017-06-16 10:00
Available from 2017-06-16 10:00

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