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Welcome to the PhosCalc wiki!
PhosCalc carries out assesment of the relative likelihoods of phosphorylation at each potentially phosphorylated S, T or Y in an amino acid sequence, based on the intensity of peaks in a corresponding mass spectrum file. The calculator is an implementation of the method used to determine the phosphorylation score for each peptide described in papers by the Mann group:
Olsen and Mann
Olsen et al
PhosCalc requires that the user provide a peptide sequence that is thought to be phosphorylated and a corresponding mass/intensity file from an MS experiment.
Broadly, PhosCalc will for each possible theoretical B and Y ion of a provided peptide calculate the mass and then look for equivalents in the mass spectrometer data and work out a likelihood for each possible phosphorylation site. The method follows closely that described here Olsen and Mann and here
Olsen et al
For more discussion please see the paper MacLean et al
If this option is selected a set of predicted water-loss ions are generated for peak matching and added to the theoretical ions for matching. For charge state = 1, dehydro = mass – 18.01057; for other charge states, dehydro = mass – (18.01057/2)
Cysteine residue mass may be selected according to any modifications in the experiment
- unmodified cysteine mass = 103.00919
- alkylated cysteine (carboxmethylation) = 103.00919 + 58.00548
- alkylated cysteine (carboxamidomethylation) = 103.00919 + 57.021464
Charge state is automatically determined from the spectrum files. A set of predicted 2+ ions will be generated if the charge state in the spectrum is 2 or higher
The hypothetical mass peaks derived from the peptide sequence are matched with the peaks from the Mass spec by allowing a window of error. This can be user-defined
Define whether the data came from an MS2 or MS3 experiment.
PhosCalc will make an educated guess on the exact phosphorylation site based on the relative scores of each of the sites. The best guess range will determine the size of the window that distinguishes the scores.