Experimental Design

Primary human renal cells were either cultured in regular medium or in   ¹³C₆-arginine (R6) and ¹³C₆¹⁵N₂-lysine (K8) medium. After 6 passages, AngII was added to SILAC-labeled cells and proteins of the treated and control cells were extracted and mixed at 1:1 protein ratio. In total, four replicates were performed, in one of which cells grown in regular medium were treated with AngII (labeling condition swapped). In the experimental settings in PEAKS Studio 8.5, control condition was specified as the reference condition so that ratios of the treated relative to control are calculated.

MS data was analyzed in PEAKS Studio 8.5 using a customized SILAC-2plex (R6, K8) method in PEAKS Q for quantification.

• Normalization of SILAC ratios in each sample

Auto normalization was first performed in PEAKS Q so that the total light and heavy intensities in each sample were equivalent since the same amount of light and heavy proteins were mixed. The adjusted normalization factor is displayed for each sample in the normalization setting window.

• Identification of differentially expressed proteins between groups

SILAC ratios of proteins were calculated using the median of peptide ratios (MS1 peak area in the labeling channel relative to the reference channel). Proteins that had significantly differential expressions between treated and control conditions across four replicates could be identified by applying a fold change filter of, e.g. at least 1.5, and a paired T-test p value smaller than 5%. Paired T-test is the statistical tool integrated in PEAKS Q for single-group SILAC data analysis.