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Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips

Nature Protocols volume 2, pages 1896–1906 (2007)Cite this article

Abstract

Mass spectrometry (MS)-based proteomics measures peptides derived from proteins by proteolytic cleavage. Before performing the analysis by matrix-assisted laser desorption/ionization–tandem mass spectrometry (MALDI–MS/MS), nanoelectrospray–MS/MS (NanoES-MS/MS) or liquid chromatography–MS/MS (LC–MS/MS), the peptide mixtures need to be cleaned, concentrated and often selectively enriched or pre-fractionated, for which we employ simple, self-made and extremely economical stop-and-go-extraction tips (StageTips). StageTips are ordinary pipette tips containing very small disks made of beads with reversed phase, cation-exchange or anion-exchange surfaces embedded in a Teflon mesh. The fixed nature of the beads allows flexible combination of disks with different surfaces to obtain multi-functional tips. Disks containing different surface functionalities and loose beads such as titania and zirconia for phosphopeptide enrichment can be combined. Incorporation into an automated workflow has also been demonstrated. Desalting and concentration takes approximately 5 min while fractionation or enrichment takes approximately 30 min.

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Figure 1: Schematic representation and production of stop-and-go-extraction tips (StageTips): panel (a) the chromatographic beads for micro-purification are embedded in a Teflon mesh and are held in place solely by the tapering of the vessel.
Figure 2: Example of a multi-stop-and-go-extraction tip (StageTip) containing a C18 disk stacked onto a strong cation exchange (SCX) disk.
Figure 3
Figure 4: Comparison of different analyses of 1.5 μg Escherichia coli lysate either directly injected for liquid chromatography-tandem mass spectrometry (LC–MS/MS) or after stop-and-go-extraction tip (StageTip) pre-fractionation into three fractions.
Figure 5: Phosphopeptide analysis using titania and zirconia following Step D.

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Acknowledgements

This work was initiated at the Center of Experimental BioInformatics (CEBI), University of Southern Denmark-Odense, supported by a generous fund of the Danish National Research Foundation. Y.I. thanks Naoyuki Sugiyama and other members for supports on phosphopeptide enrichment protocol developed in Keio University.

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Authors and Affiliations

  1. Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh, UK

    Juri Rappsilber

  2. Max Planck Institute for Biochemistry, Martinsried, Germany

    Matthias Mann

  3. Institute for Advanced Biosciences, Keio University, Yamagata, Japan

    Yasushi Ishihama

  4. PRESTO, Japan Science and Technology Agency, Tokyo, Japan

    Yasushi Ishihama

Authors
  1. Juri Rappsilber
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  2. Matthias Mann
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  3. Yasushi Ishihama
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Correspondence to Juri Rappsilber, Matthias Mann or Yasushi Ishihama.

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Rappsilber, J., Mann, M. & Ishihama, Y. Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips. Nat Protoc 2, 1896–1906 (2007). https://doi.org/10.1038/nprot.2007.261

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