Figure 1: Multi time point pSILAC mass spectrometry.

(a) Outline of the multi time point pulse-SILAC mass spectrometry experimental design. For simplification, replicate and reverse experiments are not depicted in this figure. (b) Scatter plot depicting the change of H/L protein ratios over time. Protein ratios of the 10 and 16 h time points are compared to the 6 h time point. Data in this figure are presented as means of biological replicates for T6 and T16 (n=2) or individual data points for T10 (n=1). (c) Frequencies of H/L protein ratios at different time points (data as in b). (d) Plots depicting the logarithmic H/L protein ratios over time are shown for the two validated targets CSNK1A1 and ZFP91 as well as for control proteins GAPDH, UBA1, DDB1 and CopS5. Differential turnover for CSNK1A1 and ZFP91 is observed, while stable conditions are found for controls. Data in this figure are presented as individual data points and separate r² values for the linear regression are provided for lenalidomide and DMSO samples. (e) Scatter plot comparing protein half-lives of lenalidomide treated and control samples. Protein half-lives were obtained by fitting the H/L protein ratios of T6 (n=2), T10 reverse (n=1) and T16 (n=2) time points to a decay function²⁴. For further analysis, we retained only proteins quantified in all samples and with r²>0.9 for both linear regression fits (DMSO and lenalidomide treated), resulting in a total of 2,759 proteins. CSNK1A1 was found to exhibit a reduced half-life in presence of lenalidomide. Blue and red dotted lines indicate ±3 and ±5 s.d., respectively. ZFP91 was dropped from the data analysis for missing values in two replicate measurements. The depicted delta-half-life for ZFP91 is calculated using imputed values from replicates to replace the missing values. (f) Scatter plot depicting the negative correlation between differences in protein half-life (data as in e), and differences in log₂ H/L protein ratios at the T16 time point (LENA-DMSO, mean of two biological replicates). R corresponds to the Pearson’s correlation coefficient. (g) Fold change in H/L ratios comparing lenalidomide to DMSO control treatment on the x axis (HEK293 T16 SILAC samples). Moderated t-test P values were calculated using the limma package and shown as −log₁₀ values on the y axis. The vertical dashed lines indicate ±5 s.d. log₂ fold change in H/L ratio and the horizontal dashed line indicates P value <0.001. Data shown represents two biological replicates.