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

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.