Figure 1.

Analysis of TMT ratio compression (from labeling to LC/MS/MS detection).

(A) Schematic representation assessing TMT ratio compression. Regular Caco-2, heavy Caco-2 and yeast proteins were each prepared to obtain tryptic peptides. Regular Caco-2 peptides were then loaded at indicated stoichiometric (S.) ratios, whereas heavy Caco-2 peptides with or without yeast were loaded at equal levels, followed by TMT labeling, ERLIC prefractionation and repeated LC/MS/MS analyses. The dot frames indicate sample combinations for each 6-plex TMT channel. For simplicity, the schematic displays the interference containing both heavy Caco-2 and yeast materials. (B) Schematic representation showing the mutual interference in TMT ratios between regular and heavy Caco-2 peptides. The presence of heavy Caco-2 peptides at equal theoretical ratios (R _theo) compresses the measurements of regular Caco-2 peptides from 1.05 – 1.4 R_theo to respective experimental ratios $({\tilde{R}}_{exp})$ for each TMT channel. Conversely, the presence of regular Caco-2 peptides at R_theo inflates the ratios of heavy Caco-2 peptides from R_theo to respective $(R_{exp})$for each TMT channel.^48,49 Comparisons of the experimental medians $({\tilde{R}}_{exp})$ to respective R_theo were used to calculate the compression factors (F_C) and the inflation factors (F_i)for regular and heavy Caco-2 proteins, respectively. (C) Ratio distribution of regular Caco-2 proteins under HSILACi + YEASTi (C1) or HSILACi (C2) conditions. S. ratios of regular Caco-2 proteins are indicated. (D) Respective ratio distribution of equal-level heavy Caco-2 proteins under the presence of regular Caco-2 proteins at indicated S. ratios. The vertical lines in box plots indicate the median and the whisker caps indicate the lower 10th and upper 90th percentiles, respectively. HSILACi, heavy SILAC Caco-2 interference; YEASTi, yeast interference.