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Originally published In Press as doi:10.1074/mcp.T300006-MCP200 on September 22, 2003.
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Molecular & Cellular Proteomics 2:1234-1243, 2003.
© 2003 by The American Society for Biochemistry and Molecular Biology, Inc.

Technology

Large-scale Analysis of in Vivo Phosphorylated Membrane Proteins by Immobilized Metal Ion Affinity Chromatography and Mass Spectrometry*

Thomas S. Nühse{ddagger}, Allan Stensballe§, Ole N. Jensen§,¶ and Scott C. Peck{ddagger},¶

From {ddagger} The Sainsbury Laboratory, John Innes Centre, Colney Lane, Norwich NR4 7UH, United Kingdom, and § Department of Biochemistry and Molecular Biology, University of Southern Denmark, DK-5230 Odense M, Denmark

Global analyses of protein phosphorylation require specific enrichment methods because of the typically low abundance of phosphoproteins. To date, immobilized metal ion affinity chromatography (IMAC) for phosphopeptides has shown great promise for large-scale studies, but has a reputation for poor specificity. We investigated the potential of IMAC in combination with capillary liquid chromatography coupled to tandem mass spectrometry for the identification of plasma membrane phosphoproteins of Arabidopsis. Without chemical modification of peptides, over 75% pure phosphopeptides were isolated from plasma membrane digests and detected and sequenced by mass spectrometry. We present a scheme for two-dimensional peptide separation using strong anion exchange chromatography prior to IMAC that both decreases the complexity of IMAC-purified phosphopeptides and yields a far greater coverage of monophosphorylated peptides. Among the identified sequences, six originated from different isoforms of the plasma membrane H+-ATPase and defined two previously unknown phosphorylation sites at the regulatory C terminus. The potential for large-scale identification of phosphorylation sites on plasma membrane proteins will have wide-ranging implications for research in signal transduction, cell-cell communication, and membrane transport processes.

To whom correspondence should be addressed: Scott Peck, The Sainsbury Laboratory, John Innes Centre, Colney Lane, Norwich NR4 7UH, United Kingdom. Fax: 44-(0)1603-450011; E-mail: scott.peck{at}sainsbury-laboratory.ac.uk. Ole Nørregaard Jensen, Department of Biochemistry and Molecular Biology, University of Southern Denmark, DK-5230 Odense M, Denmark. Fax: 45-6550-2467; E-mail: jenseno{at}bmb.sdu.dk.


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