To generate a y-ion filtered spectrum, one acquires two MS/MS spectra where in one case the precursor ion resolution is set to pass both ¹⁶O/¹⁸O labeled precursor ions, and in the other only the ¹⁸O precursor is allowed to undergo CID.

The scoring assigns values to all of the peaks in the ¹⁶O/¹⁸O spectrum according to the likelihood that each peak represents the first isotope of a ¹⁶O/¹⁸O doublet. In the first scoring parameter, each peak in the ¹⁶O/¹⁸O spectrum is evaluated as to whether it is the first isotope of a ¹⁶O/¹⁸O labeled fragment. Using an average amino acid composition (determined from the non-redundant database), an approximate molecular weight dependent isotope pattern is derived, which is superimposed over the ¹⁶O/¹⁸O doublet pattern. The first scoring parameter is determined by comparing these predicted isotope patterns with what was observed for each peak. The second scoring value is derived from a consideration of the relative suppression of ¹⁶O isotope peaks in an ¹⁸O-only spectrum, as compared to the ¹⁶O/¹⁸O spectrum. Once these two scoring values have been generated for each peak in the ¹⁶O/¹⁸O spectrum, the ion intensity values for each peak are multiplied by their corresponding two m/z-dependent scores. This has the effect of producing an MS/MS spectrum with enhanced intensity of C-terminal fragment ions containing the ¹⁶O isotope.