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Reproducible isolation of distinct, overlapping segments of the phosphoproteome

Nature Methods volume 4, pages 231237 (2007) | Download Citation

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Abstract

The ability to routinely analyze and quantitatively measure changes in protein phosphorylation on a proteome-wide scale is essential for biological and clinical research. We assessed the ability of three common phosphopeptide isolation methods (phosphoramidate chemistry (PAC), immobilized metal affinity chromatography (IMAC) and titanium dioxide) to reproducibly, specifically and comprehensively isolate phosphopeptides from complex mixtures. Phosphopeptides were isolated from aliquots of a tryptic digest of the cytosolic fraction of Drosophila melanogaster Kc167 cells and analyzed by liquid chromatography–electrospray ionization tandem mass spectrometry. Each method reproducibly isolated phosphopeptides. The methods, however, differed in their specificity of isolation and, notably, in the set of phosphopeptides isolated. The results suggest that the three methods detect different, partially overlapping segments of the phosphoproteome and that, at present, no single method is sufficient for a comprehensive phosphoproteome analysis.

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Acknowledgements

We thank O. Rinner, J. Malmström, P. Picotti and M. Larsen for fruitful discussions. This project has been funded in part by ETH Zurich and by federal funds from the US National Heart, Lung, and Blood Institute of the National Institutes of Health (N01-HV-28179). B.B. is the recipient of a fellowship by the Boehringer Ingelheim Fonds.

Author information

Affiliations

  1. Institute for Molecular Systems Biology, Swiss Federal Institute of Technology, ETH Hönggerberg, Wolfgang-Pauli-Str. 16, HPT, CH-8093 Zürich, Switzerland.

    • Bernd Bodenmiller
    • , Lukas N Mueller
    • , Markus Mueller
    •  & Bruno Domon
  2. Institute for Systems Biology, 1441 N. 34th Street, Seattle, Washington 98103, USA.

    • Ruedi Aebersold
  3. Institute for Molecular Systems Biology, Swiss Federal Institute of Technology, ETH Hönggerberg, Wolfgang-Pauli-Str. 16, HPT E 78, CH-8093 Zürich, Switzerland and Faculty of Natural Sciences, University of Zurich, Switzerland.

    • Ruedi Aebersold

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The authors declare no competing financial interests.

Corresponding author

Correspondence to Ruedi Aebersold.

Supplementary information

PDF files

  1. 1.

    Supplementary Fig. 1

    Amino acid usage of the identified (phospho)peptides compared to the starting material and the used protein database.

  2. 2.

    Supplementary Fig. 2

    The influence of the peptide to resin ratio on the selectivity and percentage of singly phosphorylated peptides in the case of pTiO2.

  3. 3.

    Supplementary Fig. 3

    The influence of the peptide to resin ratio on the selectivity and percentage of singly phosphorylated peptides in the case of IMAC.

  4. 4.

    Supplementary Table 1

    Values of the similarity and overlap between any two LC-MS runs as computed by Superhirn.

  5. 5.

    Supplementary Table 2

    Phosphopeptides identified with PAC, IMAC, pTiO2 and dhbTiO2.

  6. 6.

    Supplementary Table 3

    Influence of methylation prior and/or after phosphopeptide isolation using IMAC.

  7. 7.

    Supplementary Methods

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DOI

https://doi.org/10.1038/nmeth1005

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