Nonparametric mass calibration using hundreds of internal calibrants

In situations where many molecular ions (> 100) can be identified to the level of their elemental composition, such as in proteomics, metabolomics, and glycomics, a final mass calibration is possible for every sample without reliance on any analytical description of instrument behavior. This is achieved by applying a nonparametric calibration curve determined from the difference in observed, centroided m/z values of the known, internal calibrant molecular ions versus that calculated from their elemental compositions, over the m/z range. In examples here, proteomic data are examined for two sets of samples of complex mixtures composed of tryptic peptides from human and mouse blood proteins using high-resolution time-of-flight mass spectra from on-line liquid chromatography-mass spectrometry experiments. Resultant, postcalibration median absolute value mass errors and root-mean-square errors for peptides between 300 and 1100 m/z for many samples ranged from 3.1 to 4.4 and 5.2 to 6.9 ppm, respectively. The method may be applied to other types of mass spectrometers.