A method of calibrating ion collision energy used in a mass spectrometer, comprises: (a) obtaining fragment ion yield data for each of a plurality of precursor ion populations having respective mass-to-charge ratios at each of a plurality of settings of a fragmentation-energy-related variable; (b) locating, for each mass-to-charge ratio, reference values of the fragmentation-energy-related variable, each reference value corresponding to a respective reference feature of the ion yield data at the mass-to-charge ratio; (c) determining, from the plurality of locating steps, the variation, with mass-to-charge-ratio, of each of the reference values of the fragmentation-energy-related variable; (d) associating each of the reference values of the fragmentation-energy related variable with respective reference values of a dimensionless useable-fragmentation-energy variable; and (e) storing parameters describing the variation of each of the reference values of the fragmentation-energy-related variable with mass-to-charge ratio, wherein the parameters comprise coefficients of at least one non-linear equation.