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J Proteome Res. 2019 Jul 5;18(7):2706-2718. doi: 10.1021/acs.jproteome.8b00924. Epub 2019 Jun 7.

Screening a Resource of Recombinant Protein Fragments for Targeted Proteomics.

Author information

1
Science for Life Laboratory, Division of Systems Biology, Department of Protein Science , KTH-Royal Institute of Technology , SE - 171 21 Stockholm , Sweden.
2
Science for Life Laboratory, Division of Affinity Proteomics, Department of Protein Science , KTH-Royal Institute of Technology , SE - 171 21 Stockholm , Sweden.
3
Albanova University Center , KTH-Royal Institute of Technology , SE - 171 21 Stockholm , Sweden.
4
Atlas Antibodies AB , SE - 114 21 Stockholm , Sweden.
5
Department of Neuroscience - Karolinska Institute , SE - 171 65 Solna , Sweden.
6
Novo Nordisk Foundation Center for Biosustainability , Technical University of Denmark , DK - 2970 Hørsholm , Denmark.

Abstract

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, SERPINA5, 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.

KEYWORDS:

assay generation; mass spectrometry; peptide formation; protein fragment; protein quantification; recombinant proteins; spectral library; stable isotope standards; targeted proteomics; trypsin digestion

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