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Modular Stop and Go Extraction Tips with Stacked Disks for Parallel and Multidimensional Peptide Fractionation in Proteomics

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    Modular Stop and Go Extraction Tips with Stacked Disks for Parallel and Multidimensional Peptide Fractionation in Proteomics
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    Center for Experimental BioInformatics, Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, 5230 Odense M, Denmark
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    Journal of Proteome Research

    Cite this: J. Proteome Res. 2006, 5, 4, 988–994
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    https://doi.org/10.1021/pr050385q
    Published March 7, 2006
    Copyright © 2006 American Chemical Society
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    Abstract

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    Proteome complexity necessitates protein or peptide separation prior to analysis. We previously described a pipet-tip based peptide micropurification system named StageTips (STop and Go Extraction Tips), which consists of a very small disk of membrane-embedded separation material. Here, we extend this approach in several dimensions by stacking disks containing reversed phase (C18) and strong cation exchange (SCX) materials. Multidimensional fractionation as well as desalting, filtration, and concentration prior to mass spectrometry in single or tandem columns is described. C18−SCX−C18 stacked disks significantly improved protein identification by LC−MS/MS for an E. coli protein digest and by MALDI−MS for a 12 standard protein digest. Sequential fractionation based on C18- followed by SCX material was also developed. This multidimensional fractionation approach was expanded to parallel sample preparation by incorporating C18−SCX−StageTips into a 96-well plate (StagePlate). Fractions were collected into other C18−StagePlates and desalted and eluted in parallel to sample well plates or MALDI targets. This approach is suitable for high throughput protein identification for moderately complex, low abundance samples using automated nanoelectrospray−MS/MS or MALDI−MS.

    Keywords: peptide multidimensional fractionation • StageTip • microtip • SCX • C18 • proteomics • LC−MS/MS • MALDI−MS • nanoelectrospray−MS

    Copyright © 2006 American Chemical Society

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     To whom correspondence should be addressed. Y.I. Tel:  +81 29 847 7192. Fax:  +81 29 847 7614. E-mail:  [email protected]. M.M. Tel:  +49 89 8578 2557. Fax:  +49 89 8578 3209. E-mail:  [email protected].

    †

     Current address:  Laboratory of Seeds Finding Technology, Eisai Co., Ltd., Ibaraki, Japan.

    ‖

     Both authors contributed equally.

    ‡

     Current address:  The FIRC Institute for Molecular Oncology, Milan, Italy.

    §

     Current address:  Department of Proteomics and Signal Transduction, Max Planck Institute for Biochemistry, Martinsried, Germany.

    Supporting Information Available

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    Detailed procedures of StageTip fractionation for LC‐MS, MALDI, and nanoES. This material is available free of charge via the Internet at http://pubs.acs.org.

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    Cite this: J. Proteome Res. 2006, 5, 4, 988–994
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