Experiment design

Our goal was to study how phosphoproteins and gene expressions in the wild type MCF10A epithelial cell line, along with congenic TP53- or PTEN-knockout derivatives, may change in response to MMS perturbations. An overview of the experimental workflow is shown in Fig. 1, and summaries of all samples that were generated for the phosphoproteomic and genomic analyses are shown in Tables 1, ​,22.

Sample processing workflow for phosphoproteomic and RNA-seq data generation. Three congenic cell lines were paired for SILAC labeling as given in Table 1 and subsequently treated using the DNA alkylating agent methyl methanesulfonate (MMS), or mock-treated. The samples were analyzed either by RNA-seq, or were further processed for phosphoproteomic analysis by mixing the cells of the SILAC pairs, lysing the cells, and enzymatically digesting the lysates using Lys-C and trypsin. The digests were fractionated using basic (high pH) reverse phase liquid chromatography and phosphopeptides were enriched by immobilized metal affinity chromatography (IMAC). The final phosphoproteomic samples were analyzed by LC-MS/MS on an LTQ-Orbitrap Elite mass spectrometer.

Summary of the phosphoproteomic dataset results for the SILAC-labeled phosphoproteomic samples and label-swap pairs analyzed by LC-MS/MS.

The experiments indicated in bold (Expts 2-3, 4-5, and 8-9) indicate heavy and light label-swap SILAC pairs. Italic font indicates the two biological replicate experiments (Expts 1-2). Only phosphosites and phosphopeptides with phosphosite localization scores >0.8 were included in the table. For the pY, pS, and pT columns, peptides were counted in two columns if a peptide sequence contained, for example, both a pS and a pT phosphorylation; there were 2345 phosphopeptides that were counted in two columns. KO: knockout; MMS: methyl methanesulfonate-treated; WT: wild type.

Summary of RNA-seq results.

Three biological replicates were prepared for each cell line and treatment on 3 independent days. KO: knockout; MMS: methyl methanesulfonate-treated; WT: wild type.

Specifically, to allow for comparisons of phosphoprotein levels between MMS and mock treatment, and between the different cell lines, pairs of cultured cells were metabolically labeled by stable isotope labeling with amino acids in cell culture (SILAC)^12,13. In a SILAC experiment, one cell population is grown in a medium containing natural ¹²C6;¹⁴N2-lysine and ¹²C6;¹⁴N4-arginine, and another in a medium containing heavy isotopes ¹³C6;¹⁵N2-lysine and ¹³C6;¹⁵N4-arginine. When the two populations are mixed and analyzed by mass spectrometry, peptides stemming from the two populations can be distinguished by their different mass-to-charge ratios, and the relative peak intensities reflect the abundance ratios. In total, 11 pairs of samples were profiled by LC-MS/MS (Table 1).

In the RNA-seq study, three biological replicates (prepared on three independent days) were used for each cell-line and treatment group, and 18 RNA-seq profiles were generated (Table 2).