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Submitted on May 19, 2003
Revised on September 18, 2003
Accepted on September 22, 2003

Large-scale analysis of in vivo phosphorylated membrane proteins by immobilized metal ion affinity chromatography and mass spectrometry

Thomas S. Nühse, Allan Stensballe, Jensen Ole N, and Scott C. Peck

Sainsbury Laboratory, Norwich NR4 7UH

Corresponding Author: Scott.Peck{at}sainsbury-laboratory.ac.uk

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 (LC-MS/MS) 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 (SAX) 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 processe


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