Abstract
In the analysis of biological systems, it is of interest to identify the components of the system and to monitor their changes in abundance under different conditions. The AQUA (for 'absolute quantification') method allows sensitive and specific targeted quantification of protein and post-translational modifications in complex protein mixtures using stable isotope–labeled peptides as internal standards. Each AQUA experiment is composed of two stages: method development and application to a biological scenario. In the method development stage, peptides from the protein of interest are chosen and then synthesized with stable isotopes such as 13C, 2H or 15N. The abundance of these internal standards and their endogenous counterparts can be measured by mass spectrometry with selected reaction monitoring or selected ion monitoring methods. Once an AQUA method is established, it can be rapidly applied to a wide range of biological samples, from tissue culture cells to human plasma and tissue. After AQUA peptide synthesis, the development, optimization and application of AQUA analyses to a specific biological problem can be achieved in ∼1 week. Here we demonstrate the usefulness of this method by monitoring both Polo-like kinase 1 (Plk1) protein abundance in multiple lung cancer cell lines and the extent of Plk1 activation loop phosphorylation (pThr-210) during release from S phase.
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Acknowledgements
We thank S. Cullati and D. Schweppe for helpful discussion and comments on the manuscript. This work was supported by the National Institutes of Health Grant P20-RR018787 from the IDeA Program of the National Center for Research Resources and the American Cancer Society Grant IRG-82-003-24 (to S.A.G.).
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A.N.K. designed and conducted the experiments for Plk1 AQUA analysis, interpreted the data and wrote the paper; J.R. synthesized the Plk1 AQUA peptides used in this study; and S.A.G. conceived of the experiments for Plk1 AQUA analysis, interpreted the data and wrote the paper.
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Scott Gerber is a patent holder for AQUA technology, and receives royalty income from this patent. John Rush is an employee of Cell Signaling Technology, which has licensed the AQUA technology and sells AQUA peptides.
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Kettenbach, A., Rush, J. & Gerber, S. Absolute quantification of protein and post-translational modification abundance with stable isotope–labeled synthetic peptides. Nat Protoc 6, 175–186 (2011). https://doi.org/10.1038/nprot.2010.196
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DOI: https://doi.org/10.1038/nprot.2010.196
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