Protein phosphorylation regulates many cellular processes and pathways, such as cell cycle progression, signal transduction cascades and gene expression. Selective detection of phosphopeptides from proteolytic digests is a challenging and highly relevant task in many proteomics applications. Often phosphopeptides are present in small amounts and need selective isolation or enrichment before identification. Here we report a novel approach to label selectively phospho-Ser/-Thr residues by exploiting the features of a novel linear ion trap mass spectrometer. Using dansyl labelling and MS3 fragmentation, we developed a method useful for the large-scale proteomic profiling of phosphorylation sites. The new residues in the sequence were stable and easily identifiable under general conditions for tandem mass spectrometric sequencing. Copyright 2006 John Wiley & Sons, Ltd.