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Screening a Resource of Recombinant Protein Fragments for Targeted Proteomics
KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Systems Biology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Systems Biology. KTH, Centres, Science for Life Laboratory, SciLifeLab.ORCID iD: 0000-0002-5248-8568
KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Systems Biology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Systems Biology. KTH, Centres, Science for Life Laboratory, SciLifeLab.ORCID iD: 0000-0002-5388-3826
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2019 (English)In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 18, no 7, p. 2706-2718Article in journal (Refereed) Published
Abstract [en]

The availability of proteomics resources hosting protein and peptide standards, as well as the data describing their analytical performances, will continue to enhance our current capabilities to develop targeted proteomics methods for quantitative biology. This study describes the analysis of a resource of 26,840 individually purified recombinant protein fragments corresponding to more than 16,000 human protein-coding genes. The resource was screened to identify proteotypic peptides suitable for targeted proteomics efforts, and we report LC-MS/MS assay coordinates for more than 25,000 proteotypic peptides, corresponding to more than 10,000 unique proteins. Additionally, peptide formation and digestion kinetics were, for a subset of the standards, monitored using a time-course protocol involving parallel digestion of isotope-labeled recombinant protein standards and endogenous human plasma proteins. We show that the strategy by adding isotope-labeled recombinant proteins before trypsin digestion enables short digestion protocols (<= 60 min) with robust quantitative precision. In a proof-of-concept study, we quantified 23 proteins in human plasma using assay parameters defined in our study and used the standards to describe distinct clusters of individuals linked to different levels of LPA, APOE, SERPINAS, and TFRC. In summary, we describe the use and utility of a resource of recombinant proteins to identify proteotypic peptides useful for targeted proteomics assay development.

Place, publisher, year, edition, pages
American Chemical Society (ACS), 2019. Vol. 18, no 7, p. 2706-2718
Keywords [en]
targeted proteomics, stable isotope standards, mass spectrometry, protein quantification, recombinant proteins, protein fragment, trypsin digestion, spectral library, assay generation, peptide formation
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:kth:diva-255390DOI: 10.1021/acs.jproteome.8b00924ISI: 000474795500003PubMedID: 31094526Scopus ID: 2-s2.0-85067403932OAI: oai:DiVA.org:kth-255390DiVA, id: diva2:1339518
Note

QC 20190730

Available from: 2019-07-30 Created: 2019-07-30 Last updated: 2019-07-30Bibliographically approved

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Edfors, FredrikForsström, BjörnVunk, HelianFredolini, ClaudiaMaddalo, GianlucaTegel, HannaNilsson, PeterSchwenk, Jochen M.Uhlén, Mathias

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Edfors, FredrikForsström, BjörnVunk, HelianKotol, DavidFredolini, ClaudiaMaddalo, GianlucaSvensson, Anne-SophieBoström, ToveTegel, HannaNilsson, PeterSchwenk, Jochen M.Uhlén, Mathias
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Systems BiologyScience for Life Laboratory, SciLifeLabAffinity ProteomicsKTH
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