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A recombinant protein standard resource for targeted proteomics
KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab. (Uhlen)ORCID-id: 0000-0002-0017-7987
KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.ORCID-id: 0000-0002-7674-2014
KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Skolan för bioteknologi (BIO), Centra, Albanova VinnExcellence Center for Protein Technology, ProNova. Atlas Antibodies AB.
Visa övriga samt affilieringar
(Engelska)Manuskript (preprint) (Övrigt vetenskapligt)
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. 

Nationell ämneskategori
Biokemi och molekylärbiologi
Forskningsämne
Bioteknologi
Identifikatorer
URN: urn:nbn:se:kth:diva-193964OAI: oai:DiVA.org:kth-193964DiVA, id: diva2:1034918
Anmärkning

QC 20161013

Tillgänglig från: 2016-10-13 Skapad: 2016-10-13 Senast uppdaterad: 2016-10-13Bibliografiskt granskad
Ingår i avhandling
1. Targeted proteomics methods for protein quantification of human cells, tissues and blood
Öppna denna publikation i ny flik eller fönster >>Targeted proteomics methods for protein quantification of human cells, tissues and blood
2016 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
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.

Ort, förlag, år, upplaga, sidor
Stockholm: Kungliga Tekniska högskolan, 2016. s. 90
Serie
TRITA-BIO-Report, ISSN 1654-2312 ; 2016:16
Nyckelord
proteomics, mass spectrometry, protein quantification, stable isotope standard, parallel reaction monitoring, immuno-enrichment
Nationell ämneskategori
Biokemi och molekylärbiologi
Forskningsämne
Bioteknologi
Identifikatorer
urn:nbn:se:kth:diva-193951 (URN)978-91-7729-153-4 (ISBN)
Disputation
2016-11-11, Gard-aulan, Folkhälsomyndigheten, Nobels väg 18, Solna, 10:00 (Engelska)
Opponent
Handledare
Anmärkning

QC 20161013

Tillgänglig från: 2016-10-13 Skapad: 2016-10-13 Senast uppdaterad: 2016-10-14Bibliografiskt granskad

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Av författaren/redaktören
Edfors, FredrikForsström, BjörnFredolini, ClaudiaBoström, ToveMaddalo, GianlucaSvensson, Anne-SophieTegel, HannaNilsson, PeterJochen, SchwenkUhlén, Mathias
Av organisationen
Proteomik och nanobioteknologiScience for Life Laboratory, SciLifeLabAlbanova VinnExcellence Center for Protein Technology, ProNova
Biokemi och molekylärbiologi

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