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Investigating the correlation of protein and mRNA levels in human cell lines using quantitative proteomics and transcriptomics
KTH, School of Biotechnology (BIO), Protein Technology.
KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. (Proteomics)ORCID iD: 0000-0002-7692-1100
KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. (Proteomics)ORCID iD: 0000-0001-7034-0850
Karlinska Institute, Cancer Proteomics Mass Spectrometry, Dep. of Oncology-Pathology.
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

An important topic of discussion in proteomics is the degree of correlation of RNA and protein levels in cells, tissues and organs. In this study, the difference in protein and mRNA levels for a number of selected gene targets were investigated across six human cell lines using quantitative proteomics and next generation sequencing-based transcriptomics. The copy numbers of 32 proteins were determined using an absolute quantitative proteomics approach (PrEST-SILAC), where heavy isotope-labeled protein fragments were used as internal standards. A cross evaluation of protein copy numbers determined by mass spectrometry and staining profiles using immunohistochemistry showed good correlation. The mRNA levels were determined using RNA sequencing based on digital counting of sequencing reads and the levels determined as FPKM values. Comparison of the relative variations in mRNA and protein levels for individual genes across the six cell lines showed correlation between protein and mRNA levels, including six genes with high variability in expression levels in the six cell lines resulting in an average correlation of 0.9 (Spearman's rank coefficient). In summary, the analysis of the selected protein targets supports the conclusion that the translation rate across cell lines correlates for a particular gene, suggesting that individual protein levels can be predicted from the respective mRNA levels by defining the relation between protein and mRNA, specific for each human gene.

National Category
Natural Sciences
Research subject
Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-154507OAI: oai:DiVA.org:kth-154507DiVA: diva2:757207
Note

QS 2015

Available from: 2014-10-21 Created: 2014-10-21 Last updated: 2015-02-17Bibliographically approved
In thesis
1. High-throughput protein analysis using mass spectrometry-based methods
Open this publication in new window or tab >>High-throughput protein analysis using mass spectrometry-based methods
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

In the field of proteomics, proteins are analyzed and quantified in high numbers. Protein analysis is of great importance and can for example generate information regarding protein function and involvement in disease. Different strategies for protein analysis and quan- tification have emerged, suitable for different applications. The focus of this thesis lies on protein identification and quantification using different setups and method development has a central role in all included papers.

The presented research can be divided into three parts. Part one describes the develop- ment of two different screening methods for His6-tagged recombinant protein fragments. In the first investigation, proteins were purified using immobilized metal ion affinity chro- matography in a 96-well plate format and in the second investigation this was downscaled to nanoliter-scale using the miniaturized sample preparation platform, integrated selective enrichment target (ISET). The aim of these investigations was to develop methods that could work as an initial screening step in high-throughput protein production projects, such as the Human Protein Atlas (HPA) project, for more efficient protein production and purification. In the second part of the thesis, focus lies on quantitative proteomics. Protein fragments were produced with incorporated heavy isotope-labeled amino acids and used as internal standards in absolute protein quantification mass spectrometry experiments. The aim of this investigation was to compare the protein levels obtained using quanti- tative mass spectrometry to mRNA levels obtained by RNA sequencing. Expression of 32 different proteins was studied in six different cell lines and a clear correlation between protein and mRNA levels was observed when analyzing genes on an individual level. The third part of the thesis involves the antibodies generated within the HPA project. In the first investigation a method for validation of antibodies using protein immunoenrichment coupled to mass spectrometry was described. In a second study, a method was developed where antibodies were used to capture tryptic peptides from a digested cell lysate with spiked in heavy isotope-labeled protein fragments, enabling quantification of 20 proteins in a multiplex format. Taken together, the presented research has expanded the pro- teomics toolbox in terms of available methods for protein analysis and quantification in a high-throughput format.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2014. x, 121 p.
Series
TRITA-BIO-Report, ISSN 1654-2312 ; 2014:15
Keyword
Proteomics, mass spectrometry, affinity proteomics, immunoenrichment, immunoprecipitation, IMAC, screening, protein production, protein purification, ISET, quantification, SILAC, stable isotope standard, antibody validation
National Category
Natural Sciences
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-154513 (URN)978-91-7595-292-5 (ISBN)
Public defence
2014-11-14, F3, Lindstedtsvägen 26, KTH, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

QC 20141022

Available from: 2014-10-22 Created: 2014-10-21 Last updated: 2015-02-17Bibliographically approved

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Lundberg, EmmaTegel, HannaUhlén, MathiasHober, Sophia

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