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Fast and easy phosphopeptide fractionation by combinatorial ERLIC-SCX solid-phase extraction for in-depth phosphoproteome analysis

Nature Protocols volume 11, pages 37–45 (2016)Cite this article

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Abstract

Mass spectrometry–based phosphoproteomic analysis is a powerful method for gaining a global, unbiased understanding of cellular signaling. Its accuracy and comprehensiveness stands or falls with the quality and choice of the applied phosphopeptide prefractionation strategy. This protocol covers a powerful but simple and rapid strategy for phosphopeptide prefractionation. The combinatorial use of two distinct chromatographic techniques that address the inverse physicochemical properties of peptides allows for superior fractionation efficiency of multiple phosphorylated peptides. In the first step, multiphosphorylated peptides are separated according to the number of negatively charged phosphosites by electrostatic repulsion-hydrophilic interaction chromatography (ERLIC). A subsequent strong cation exchange (SCX) step separates mostly singly phosphorylated peptides in the ERLIC flow-through according to their positive charge. The presented strategy is inexpensive and adaptable to large and small amounts of starting material, and it allows highly multiplexed sample preparation. Because of its implementation as solid-phase extraction, the entire workflow takes only 2 h to complete.

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Figure 1: Diagram illustrating the workflow of the protocol.
Figure 2: Illustration of the SPE setup for ERLIC and SCX.
Figure 3: Average solution net charges of identified peptides in each fractionation method.
Figure 4: Distributions of identified phosphopeptides by SPE of ERLIC (E1–E5) and SCX (FTS1–S5) fractionation methods.
Figure 5: Distributions of amino acids neighboring phosphorylation sites.

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Acknowledgements

The research leading to these results has received funding from the Excellence Initiative of the German Federal and State Governments through FRIAS and BIOSS, from the German Research Foundation (DFG) via SFB1140, KIDGEM Project C02, and project DE 1757/2-1. We thank Eksigent/AB Sciex for the generous gift of a 2D nanoLC and R. van Soest for his technical advice on maintenance and troubleshooting.

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Author notes

  1. Mostafa Zarei

    Present address: Present address: Solvias AG, Kaiseraugst, Switzerland.,

  2. Mostafa Zarei and Adrian Sprenger: These authors contributed equally to this work.

Authors and Affiliations

  1. Freiburg Institute for Advanced Studies (FRIAS), University of Freiburg, Freiburg, Germany

    Mostafa Zarei, Adrian Sprenger, Michal Rackiewicz & Joern Dengjel

  2. Center for Biological Systems Analysis (ZBSA), University of Freiburg, Freiburg, Germany

    Mostafa Zarei, Adrian Sprenger, Michal Rackiewicz & Joern Dengjel

  3. Department of Dermatology, Medical Center, University of Freiburg, Freiburg, Germany

    Adrian Sprenger, Michal Rackiewicz & Joern Dengjel

  4. BIOSS Centre for Biological Signalling Studies, University of Freiburg, Freiburg, Germany

    Adrian Sprenger & Joern Dengjel

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  1. Mostafa Zarei
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Contributions

M.Z. and A.S. set up the protocol and performed the experiments. M.R. performed data analysis. J.D. wrote the manuscript with input from all authors.

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Correspondence to Joern Dengjel.

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The authors declare no competing financial interests.

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Zarei, M., Sprenger, A., Rackiewicz, M. et al. Fast and easy phosphopeptide fractionation by combinatorial ERLIC-SCX solid-phase extraction for in-depth phosphoproteome analysis. Nat Protoc 11, 37–45 (2016). https://doi.org/10.1038/nprot.2015.134

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