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A recombinant protein standard resource for targeted proteomics
KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab. (Uhlen)ORCID iD: 0000-0002-0017-7987
KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.ORCID iD: 0000-0002-7674-2014
KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova. Atlas Antibodies AB.
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Here, we have used a resource of 26,000 recombinant protein fragments to create custom libraries of standards for targeted proteomics based on parallel reaction monitoring (PRM). The recombinant fragments can be produced in a bacterial cell factory to generate heavy isotope labeled standards for absolute quantification of the corresponding protein targets and be used to produce high- quality spectral libraries. Altogether, coordinates for 25,684 unique proteotypic peptide assays have been experimentally defined covering 10,163 human proteins. The protocol allows for precise monitoring of digestion kinetics and thus enables to select peptides that behave quantitative during the sample preparation process. We show that the quantification tag of each recombinant protein fragment can be used for accurate retention time prediction and allows for assay standardization across different method parameters. The use of this resource was illustrated by determining the absolute concentrations of selected protein targets using multiplex targeted proteomics assays for determination of quantitative assessment of 49 protein targets in serum samples. 

National Category
Biochemistry and Molecular Biology
Research subject
Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-193964OAI: oai:DiVA.org:kth-193964DiVA: diva2:1034918
Note

QC 20161013

Available from: 2016-10-13 Created: 2016-10-13 Last updated: 2016-10-13Bibliographically approved
In thesis
1. Targeted proteomics methods for protein quantification of human cells, tissues and blood
Open this publication in new window or tab >>Targeted proteomics methods for protein quantification of human cells, tissues and blood
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The common concept in this thesis was to adapt and develop quantitative mass spectrometric assays focusing on reagents originating from the Human Protein Atlas project to quantify proteins in human cell lines, tissues and blood. The work is based around stable isotope labeled protein fragment standards that each represent a small part of a human protein-coding gene. This thesis shows how they can be used in various formats to describe the protein landscape and be used to standardize mass spectrometry experiments. The first part of the thesis describes the use of antibodies in combination with heavy stable isotope labeled antigens to establish a semi-automated protocol for protein quantification of complex samples with fast analysis time  (Paper~I). Paper II introduces a semi-automated cloning protocol that can be used to selectively clone variants of recombinant proteins, and highlights the automation process that is necessary for large-scale proteomics endeavors. This paper also describes the technology that was used to clone all protein standards that are used in all of the included papers.

                     

The second part of the thesis includes papers that focus on the generation and application of antibody-free targeted mass spectrometry methods. Here, absolute protein copy numbers were determined across human cell lines and tissues (Paper III) and the protein data was correlated against transcriptomics data. Proteins were quantified to validate antibodies in a novel method that evaluates antibodies based on differential protein expression across multiple cell lines (Paper IV). Finally, a large-scale study was performed to generate targeted proteomics assays (Paper V) based on protein fragments. Here, assay coordinates were mapped for more than 10,000 human protein-coding genes and a subset of peptides was thereafter used to determine absolute protein levels of 49 proteins in human serum.

                     

In conclusion, this thesis describes the development of methods for protein quantification by targeted mass spectrometry and the use of recombinant protein fragment standards as the common denominator.

Place, publisher, year, edition, pages
Stockholm: Kungliga Tekniska högskolan, 2016. 90 p.
Series
TRITA-BIO-Report, ISSN 1654-2312 ; 2016:16
Keyword
proteomics, mass spectrometry, protein quantification, stable isotope standard, parallel reaction monitoring, immuno-enrichment
National Category
Biochemistry and Molecular Biology
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-193951 (URN)978-91-7729-153-4 (ISBN)
Public defence
2016-11-11, Gard-aulan, Folkhälsomyndigheten, Nobels väg 18, Solna, 10:00 (English)
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Supervisors
Note

QC 20161013

Available from: 2016-10-13 Created: 2016-10-13 Last updated: 2016-10-14Bibliographically approved

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Edfors, FredrikForsström, BjörnFredolini, ClaudiaBoström, ToveMaddalo, GianlucaSvensson, Anne-SophieTegel, HannaNilsson, PeterJochen, SchwenkUhlén, Mathias
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