Fig. 4.

Label-swap replica expose quantitation error in fully automated quantitation. A, 103 cross-linked peptides from the model data set can be divided into four subgroups based on fully automated MaxQuant quantitation. B, Cross-linked peptides that were consistently quantified in label-swap replica showed good agreement on both signal type assignment and C3b/C3 signal ratios between the MaxQuant quantitation and the reference data set for 159 corresponding quantitation readout. C, Implying from the MS signal of cross-linked peptide LES(cl)EETMoxVLEAHDAQGDVPVTVTVHDFPGK - IFTVNHK(cl)LLPVGR, it was mis-quantified as a C3 unique signal in the forward-labeled analysis (C3-light; C3b-heavy) because of highly overlapped isotope envelopes of the light and heavy signals, and in the reverse-labeled analysis (C3-heavy, C3b-light) due incomplete isotope envelope for the light signal. D, All five cross-linked peptides that were mis-classified as singlet signals in both label-swap replicas showed larger overlap between their light and heavy signals in respect to correctly quantified doublet cross-linked peptides. The signal overlap factor is calculated as the mass of the cross-linked peptide divided by the mass difference between the light and heavy signals. E, Comparison of MaxQuant quantitation readout against the reference data set on signal type assignments and C3b/C3 signal ratios for 11 cross-linked peptides that were quantified in only one of the label-swap replicas.