Figure 1.

Illustration of the attributes considered in the non-outlier fragment ion (NOFI) ranking algorithm and visualization of the NOFI concept. A) The attributes related to chromatographic properties of the peptide are the retention time deviation (RTd) and the full width at half maximum deviation of the LC peak (FWHMd), as explained in Methods. The library intensity rank corresponds to the red circles, assuming that the SWATH intensities have the same fragment ion order as the DDA library intensities. The NOFI rank corresponds to the green circles, in which the fragment ion with deviated attribute values is pushed down to lower priority ranks. B) The attributes related to the fragmentation pattern of the peptide (i.e. IRd and IRrep) are computed for each fragment ion from the intensity ratio (IR_j) between the SWATH and the DDA library spectra. Top panel: in the absence of interferences the relative intensities show the same pattern and the ratio of the absolute intensities is ideally constant (the absolute intensities in the SWATH spectrum have twice the values of the library spectrum, e.g. 5000 a.u. in SWATH compared to 2500 a.u. in DDA for the most intense fragment ion). Bottom panel: in the presence of interferences (grey ellipses), the intensity ratios of interference-affected fragment ions are deviated from the remaining fragment ions of the peptide. C) Left panel: vectors combining the four attributes represent the fragment ions of a peptide. Right panel: considering a projection on the first two dimensions (RTd and FWHMd attributes), high priority ranks are assigned to the fragment ions that are less deviated (closer to the center) and the deviated fragment ions are assigned lower priority ranks.