title: A Protein Epitope Signature Tag (PrEST) library allows SILAC-based absolute quantification and multiplexed determination of protein copy numbers in cell lines.
abstract: Mass spectrometry-based proteomics increasingly relies on relative or absolute quantification. In relative quantification, stable isotope based methods often allow mixing at early stages of sample preparation, whereas for absolute quantification this has generally required recombinant expression of full length, labeled protein standards. Here we make use of a very large library of Protein Epitope Signature Tags (PrESTs) that has been developed in the course of the Human Protein Atlas Project. These PrESTs are expressed recombinantly in E. coli and they consist of a short and unique region of the protein of interest as well as purification and solubility tags. We first quantify a highly purified, stable isotope labeling of amino acids in cell culture (SILAC)-labeled version of the solubility tag and use it determine the precise amount of each PrEST by its SILAC ratios. The PrESTs are then spiked into cell lysates and the SILAC ratios of PrEST peptides to peptides from endogenous target proteins yield their cellular quantities. The procedure can readily be multiplexed, as we demonstrate by simultaneously determining the copy number of 40 proteins in HeLa cells. Among the proteins analyzed, the cytoskeletal protein vimentin was found to be most abundant with 20 million copies per cell, while the transcription factor and oncogene FOS only had 6000 copies. Direct quantification of the absolute amount of single proteins is possible via a SILAC experiment in which labeled cell lysate is mixed both with the heavy labeled solubility tag and with the corresponding PrEST. The SILAC-PrEST combination allows accurate and streamlined quantification of the absolute or relative amount of proteins of interest in a wide variety of applications.
authors.affiliation: Department of Proteomics and Signal Transduction, Max-Planck Institute of Biochemistry, D-82152 Martinsried, Germany.
authors.forename: Marlis
authors.initials: M
authors.lastname: Zeiler
authors.forename: Werner L
authors.initials: WL
authors.lastname: Straube
authors.forename: Emma
authors.initials: E
authors.lastname: Lundberg
authors.forename: Mathias
authors.initials: M
authors.lastname: Uhlen
authors.forename: Matthias
authors.initials: M
authors.lastname: Mann
mesh_terms: D002853:Chromatography, Liquid; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000032:analysis* / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D007553:Isotope Labeling; D013058:Mass Spectrometry; D010446:Peptide Fragments / Q000032:analysis / Q000378:metabolism; D011506:Proteins / Q000032:analysis* / Q000378:metabolism*; D040901:Proteomics; D011994:Recombinant Proteins / Q000378:metabolism
chemical_list: D000939:Epitopes; D010446:Peptide Fragments; D011506:Proteins; D011994:Recombinant Proteins