Molecular & Cellular Proteomics · 2003 · 543 citations · 45 references
Life SciencesBiochemistryMolecular BiologyMass Spectrometric AnalysisProtein BiochemistryProteomicsMass SpectrometryMass Spectrometry-based ProteomicsPeptide AnalysisPeptide IdentificationProtein IdentificationProtein SeparationCapillary Liquid ChromatographyImmobilized Metal Affinity ChromatographyMass Spectrometry ProteomicsMembrane Protein AnalysisPeptide Mass SpectrometryPeptide SeparationPhosphopeptide IdentificationProtein Separation Techniques
Protein phosphorylation studies require enrichment due to the low abundance of phosphoproteins, and while IMAC has shown promise for large‑scale phosphopeptide analysis, it is often criticized for poor specificity. The study aimed to evaluate IMAC coupled with capillary LC‑MS for identifying Arabidopsis plasma membrane phosphoproteins and to develop a two‑dimensional separation scheme using strong anion exchange before IMAC to reduce complexity and enhance monophosphopeptide coverage. They employed IMAC combined with capillary liquid chromatography‑tandem mass spectrometry, preceded by strong anion exchange chromatography, to isolate and sequence phosphopeptides from plasma membrane digests. The approach yielded >75 % pure phosphopeptides, identified six H⁺‑ATPase isoforms with two novel regulatory phosphorylation sites, and demonstrates that large‑scale phosphoproteome mapping of plasma membrane proteins can advance studies of signal transduction, cell‑cell communication, and transport.
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.
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Metal chelate affinity chromatography, a new approach to protein fractionation
Jerker Porath, Jan Carlsson, Ingmar Olsson et al. · Nature · 1975 · 2.1K citations
Miguel Ángel Medina Torres, Jeffery L. Dangl, Jonathan D. G. Jones · Proceedings of the National Academy of Sciences · 2001 · 1.7K citations · Full text