Multiple reaction monitoring to identify sites of protein phosphorylation with high sensitivity.

Phosphorylation governs the activity of many proteins. Insight into molecular mechanisms in biology would be immensely improved by robust, sensitive methods for identifying precisely sites of phosphate addition. An approach to selective mapping of protein phosphorylation sites on a specific target protein of interest using LC-MS is described here. In this approach multiple reaction monitoring is used as an extremely sensitive MS survey scan for potential phosphopeptides from a known protein. This is automatically followed by peptide sequencing and subsequent location of the phosphorylation site; both of these steps occur in a single LC-MS run, providing greater efficiency of sample use. The method is capable of detecting and sequencing phosphopeptides at low femtomole levels with high selectivity. As proof of the value of this approach in an experimental setting, a key Schizosaccharomyces pombe cell cycle regulatory protein, Cyclin B, was purified, and associated proteins were identified. Phosphorylation sites on these proteins were located. The technique, which we have called multiple reaction monitoring-initiated detection and sequencing (MIDAS), is shown to be a highly sensitive approach to the determination of protein phosphorylation.