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Rapid empirical discovery of optimal peptides for targeted proteomics


We report a method for high-throughput, cost-efficient empirical discovery of optimal proteotypic peptides and fragment ions for targeted proteomics applications using in vitro–synthesized proteins. We demonstrate the approach using human transcription factors, which are typically difficult, low-abundance targets and empirically derived proteotypic peptides for 98% of the target proteins. We show that targeted proteomic assays developed using our approach facilitate robust in vivo quantification of human transcription factors.

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Figure 1: Development of targeted proteomics assays using enriched in vitro–synthesized full-length proteins.
Figure 2: Targeted assays can be efficiently developed using in vitro–synthesized proteins and applied to measure proteins in vivo.


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We thank P. von Haller, M. Bereman, E. Hommema and J. Rogers for their discussions and technical assistance. This work was supported in part by the University of Washington's Proteomics Resource (UWPR95794), the Thermo Scientific Pierce Human In vitro Translation Research Grant, and US National Institutes of Health grants P41RR011823 (M.J.M.) and U54HG004592 (J.A.S.).

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A.B.S., J.A.S. and M.J.M. conceived and designed the experiments, and wrote the paper. A.B.S. and K.L. performed the wet laboratory experiments. A.B.S. and B.M. analyzed the data.

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Correspondence to John A Stamatoyannopoulos or Michael J MacCoss.

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Competing interests

The authors received financial support from ThermoFisher Scientific.

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Supplementary Figures 1–7, Supplementary Data 1–2, Supplementary Note 1 (PDF 4072 kb)

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Stergachis, A., MacLean, B., Lee, K. et al. Rapid empirical discovery of optimal peptides for targeted proteomics. Nat Methods 8, 1041–1043 (2011).

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