SP19A Phosphoprotein profiling by negative mode precursor ion scanning

An important goal in cancer research is to monitor phosphoprotein changes in order to identify downstream targets of dysregulated signaling pathways. We used a precursor ion scanning approach described by Carr et al,¹ which identifies phosphopeptides in negative ion mode by their loss of a −79-Da signature ion (PO_3⁻). We first compared three methods for phosphopeptide detection in the protein kinase, Mps1. Using a 4000 QTrap mass spectrometer, standard analysis by LC/MS/MS in positive mode identified 27 phosphopeptides containing 22 phosphosites. Precursor ion scanning in negative mode using the same instrument identified 47 phosphopeptides containing 34 phosphosites, with detection sensitivity ~10 fmol. Using a LTQ-Orbitrap mass spectrometer, MS³ on peptide ions that underwent neutral loss of H₃PO₄ during MS/MS identified 30 phosphopeptides and 28 phosphosites. Thus, precursor ion scanning showed the highest performance in identifying phosphopeptides in simple mixtures.Next, we examined human melanoma cells treated with and without U0126, a drug that inhibits the constitutively activated B-Raf/MAPK pathway. Cytosolic proteins were resolved by SAX-FPLC, and proteins in each fraction were proteolyzed. Peptides in each fraction were separated by nanoflow RP-HPLC and phosphopeptides monitored by precursor ion scanning, triggering MS/MS upon detection of the −79-Da signature ion. In parallel, peptides were analyzed by positive mode LC/MS/MS in order to monitor protein abundance changes by spectral counting. In-house algorithms utilizing OpenMS modules were developed to detect phosphopeptide peaks, match them to MS/MS spectra, group peaks over consecutive fractions, and quantify and sum intensities. More than 20,000 peaks could be detected over all SAX fractions, representing ~5000 grouped phosphopeptide candidates. About 350 phosphopeptides were manually validated, of which ~10% were responsive to drug treatment. Thus, targets of dysregulated B-Raf/MAPK signaling in melanoma can be identified using precursor ion scanning and detection of phosphopeptides in complex samples.