Nanoelectrospray‐based detection and sequencing of substoichiometric amounts of phosphopeptides in complex mixtures

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Abstract
A novel immobilized metal affinity chromatography (IMAC)‐based protocol was successfully used for sample preparation prior to nanoelectrospray‐based sequencing of phosphopeptides. In a first step, phosphorylated peptides are detected in an unseparated peptide mixture using precursor ion scanning in the negative ion mode on a triple‐quadrupole mass spectrometer. Then the phosphorylated peptides are enriched by passing the sample over an IMAC column and sequenced in the positive ion mode. The IMAC‐based enrichment allows the sequencing of phosphorylated peptides even if other, much more abundant, peptides are present at the same m/z value in the original mixture. Using this two‐step approach, we are able to combine the simplicity, sensitivity and specificity of precursor ion scanning‐based detection of phosphopeptides using the nanoelectrospray ion source with the ability to sequence phosphorylated peptides even if they are present in substoichiometric amounts. Copyright © 2003 John Wiley & Sons, Ltd.
Funding Information
  • BMBF (BioFuture 0311862.)

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