An extra dimension in protein tagging by quantifying universal proteotypic peptides using targeted proteomics.
The use of protein tagging to facilitate detailed characterization of target proteins has not only revolutionized cell biology, but also enabled biochemical analysis through efficient recovery of the protein complexes wherein the tagged proteins reside. The endogenous use of these tags for detailed protein characterization is widespread in lower organisms that allow for efficient homologous recombination. With the recent advances in genome engineering, tagging of endogenous proteins is now within reach for most experimental systems, including mammalian cell lines cultures. In this work, we describe the selection of peptides with ideal mass spectrometry characteristics for use in quantification of tagged proteins using targeted proteomics. We mined the proteome of the hyperthermophile Pyrococcus furiosus to obtain two peptides that are unique in the proteomes of all known model organisms (proteotypic) and allow sensitive quantification of target proteins in a complex background. By combining these 'Proteotypic peptides for Quantification by SRM' (PQS peptides) with epitope tags, we demonstrate their use in co-immunoprecipitation experiments upon transfection of protein pairs, or after introduction of these tags in the endogenous proteins through genome engineering. Endogenous protein tagging for absolute quantification provides a powerful extra dimension to protein analysis, allowing the detailed characterization of endogenous proteins.
VIB Medical Biotechnology Center, B-9000 Ghent, Belgium.|Department of Biochemistry, Ghent University, B-9000 Ghent, Belgium.
Giel
G
Vandemoortele
VIB Medical Biotechnology Center, B-9000 Ghent, Belgium.|Department of Biochemistry, Ghent University, B-9000 Ghent, Belgium.
An
A
Staes
VIB Medical Biotechnology Center, B-9000 Ghent, Belgium.|Department of Biochemistry, Ghent University, B-9000 Ghent, Belgium.
Giulia
G
Gonnelli
VIB Medical Biotechnology Center, B-9000 Ghent, Belgium.|Department of Biochemistry, Ghent University, B-9000 Ghent, Belgium.
Noortje
N
Samyn
VIB Medical Biotechnology Center, B-9000 Ghent, Belgium.|Department of Biochemistry, Ghent University, B-9000 Ghent, Belgium.
Delphine
D
De Sutter
VIB Medical Biotechnology Center, B-9000 Ghent, Belgium.|Department of Biochemistry, Ghent University, B-9000 Ghent, Belgium.
Elien
E
Vandermarliere
VIB Medical Biotechnology Center, B-9000 Ghent, Belgium.|Department of Biochemistry, Ghent University, B-9000 Ghent, Belgium.
Evy
E
Timmerman
VIB Medical Biotechnology Center, B-9000 Ghent, Belgium.|Department of Biochemistry, Ghent University, B-9000 Ghent, Belgium.
Kris
K
Gevaert
VIB Medical Biotechnology Center, B-9000 Ghent, Belgium.|Department of Biochemistry, Ghent University, B-9000 Ghent, Belgium.
Lennart
L
Martens
VIB Medical Biotechnology Center, B-9000 Ghent, Belgium.|Department of Biochemistry, Ghent University, B-9000 Ghent, Belgium.
Sven
S
Eyckerman
D019843:Archaeal Proteins / Q000737:chemistry / Q000378:metabolism*; D003198:Computer Simulation; D045325:HCT116 Cells; D006801:Humans; D010455:Peptides / Q000302:isolation & purification*; D060066:Protein Interaction Maps; D040901:Proteomics / Q000379:methods*; D020027:Pyrococcus furiosus / Q000378:metabolism*
D019843:Archaeal Proteins; D010455:Peptides