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A Protein Epitope Signature Tag (PrEST) library allows SILAC-based absolute quantification and multiplexed determination of protein copy numbers in cell lines.

Zeiler M, Straube WL, Lundberg E, Uhlen M, Mann M - Mol. Cell Proteomics (2011)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Proteomics and Signal Transduction, Max-Planck Institute of Biochemistry, D-82152 Martinsried, Germany.

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.

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Accuracy of ABP quantification.A, Density plot of the overall distribution of the 43 coefficients of variation (CVs) of the ABP peptides measured on a benchtop Exactive mass spectrometer. B, Representative example proteins showing the H/L peptide ratios of the ABP peptides deriving from the ABP standard and the ABP peptides in the PrESTs and their coefficients of variation (CVs).
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Figure 2: Accuracy of ABP quantification.A, Density plot of the overall distribution of the 43 coefficients of variation (CVs) of the ABP peptides measured on a benchtop Exactive mass spectrometer. B, Representative example proteins showing the H/L peptide ratios of the ABP peptides deriving from the ABP standard and the ABP peptides in the PrESTs and their coefficients of variation (CVs).

Mentions: Typically, at least eight labeled ABP peptides could be quantified against the corresponding ABP peptides from the PrESTs, leading to a median coefficient of variation (CV) of 7% for PrEST quantification (Fig. 2A).


A Protein Epitope Signature Tag (PrEST) library allows SILAC-based absolute quantification and multiplexed determination of protein copy numbers in cell lines.

Zeiler M, Straube WL, Lundberg E, Uhlen M, Mann M - Mol. Cell Proteomics (2011)

Accuracy of ABP quantification.A, Density plot of the overall distribution of the 43 coefficients of variation (CVs) of the ABP peptides measured on a benchtop Exactive mass spectrometer. B, Representative example proteins showing the H/L peptide ratios of the ABP peptides deriving from the ABP standard and the ABP peptides in the PrESTs and their coefficients of variation (CVs).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3316735&req=5

Figure 2: Accuracy of ABP quantification.A, Density plot of the overall distribution of the 43 coefficients of variation (CVs) of the ABP peptides measured on a benchtop Exactive mass spectrometer. B, Representative example proteins showing the H/L peptide ratios of the ABP peptides deriving from the ABP standard and the ABP peptides in the PrESTs and their coefficients of variation (CVs).
Mentions: Typically, at least eight labeled ABP peptides could be quantified against the corresponding ABP peptides from the PrESTs, leading to a median coefficient of variation (CV) of 7% for PrEST quantification (Fig. 2A).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Proteomics and Signal Transduction, Max-Planck Institute of Biochemistry, D-82152 Martinsried, Germany.

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.

Show MeSH
Related in: MedlinePlus