Abstract
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.
Highlights
Phosphorylation governs the activity of many proteins
We demonstrate below how this relatively simple approach can enhance the development of phosphoproteomics for the biologist
Further experiments were performed to assess whether monitoring-initiated detection and sequencing (MIDAS) can identify phosphorylation sites on specific proteins in a simple mixture such as that obtained from a conventional one-dimensional gel band of an immunoprecipitate
Summary
Preparation of Samples—[Glu]Fibrinopeptide B (Sigma) was diluted using 2% (v/v) acetonitrile, 0.1% (v/v) formic acid and spiked at appropriate levels as a control compound. Bovine ␣-casein S1 and S2 (Sigma) was resuspended at a known concentration in 25 mM ammonium bicarbonate and digested by addition of modified porcine trypsin (Promega, Southampton, UK) at a 50:1 protein:enzyme ratio and incubated at 37 °C overnight This provided a stock concentration of 20 pmol lϪ1 that was further diluted and mixed using 2% (v/v) acetonitrile, 0.1% (v/v) formic acid to provide working standards. Transitions were included for all tryptic peptides (maximum one missed cleavage) containing Ser, Thr, or Tyr residues with either one or two modifications and for doubly and triply charged species for the Q1 mass range 400 –1600 m/z. The number of MRMs is dependent on various factors, including protein size, number of peptides following digestion, and the number of potential phosphorylation sites It is, important to optimize the cycle time to around or below 10 s. Data were examined manually using the Applied Biosystems BioExplore feature of the Analyst 1.4 software package
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