Aberrant phospho-signaling is a hallmark of cancer. We investigated kinase-substrate regulation of 33,239 phosphorylation sites (phosphosites) in 77 breast tumors and 24 breast cancer xenografts. Our search discovered 2,134 quantitatively-correlated kinase-phosphosite pairs, enriching for and extending experimental or binding-motif predictions. Among the 91 kinases with auto-phosphorylation, elevated EGFR, ERBB2, PRKG1, and WNK1 regulations were enriched in basal, HER2-E, Luminal A, and Luminal B breast cancers, respectively, revealing subtype-specific signaling. CDKs, MAPKs, and ataxia-telangiectasia proteins were dominant, master regulators of substrate-phosphorylation, whose activities are not captured by genomic evidence. We unveiled phospho-signaling and druggable targets from 113 kinase-substrate pairs and cascades downstream of kinases, including AKT1, BRAF and EGFR. We further identified kinase-substrate-pairs associated with clinical or immune signatures and experimentally validated activated phosphosites of ERBB2, EIF4EBP1, and EGFR. Overall, kinase-substrate regulation revealed by the largest unbiased global phosphorylation data to date connects driver events to their signaling effects.

SRM Validation Stable Isotope-Labeled Peptides Crude stable isotope-labeled peptides (SI peptides) were synthesized with 13C/15N on C-terminal lysine or arginine (New England Peptide, Gardner, MA). The SI peptides were dissolved individually in 15% acetonitrile (ACN) and 0.1% formic acid (FA) at a concentration of 1.5 mM and stored at −80 °C. A mixture of these SI peptides was made with a final concentration of 10 pmol/μL for each peptide. SRM Assay Development To evaluate the peptide quality and select the best responsive transitions for each peptide, 500 fmol/µL of heavy peptide mixtures were subjected to high-resolution mass spectrometry (MS) analysis using the Orbitrap Fusion Lumos instrument (Thermo Fisher Scientific, San Jose, CA) running in the HCD mode40. The resulting RAW files were processed with DTARefinery 41 and MS-GF+42,43 to match against the RefSeq human protein sequence database, released on April 18, 2017 (46,174 proteins) for generating a list of MS/MS fragment ions derived from SI peptides. The 6 most intensive fragment ions for each peptide were initially selected based on corresponding MS/MS spectra. The collision energies for individual transitions were obtained by using empirical equations from the Skyline software44. Secondly, LC-SRM was used to further evaluate all heavy peptides for the LC performance (e.g., the stability of peptide retention time), MS response (e.g., reliable heavy peptides identification), transition interferences, and endogenous peptide detectability by spiking them into water and the pooled wild type samples. In the end, 3 or more transitions per peptide were selected for configuration of the final panel of assays for reproducible targeted quantification. Phosphopeptides enrichment by IMAC Two hundred micrograms of tryptic peptides spiked with 1000 fmol of each of the crude SI peptides were subjected to phosphopeptides enrichment via immobilized metal affinity chromatography (IMAC)45,46. The in-house-made IMAC tip was capped in a tip-end with a 20-μm polypropylene frits disk followed by packing with Ni-NTA silica resin (QIAGEN, Hilden, Germany). First, Ni2+ ions were removed by adding 50 mM EDTA in 1 M NaCl. The tip was then activated with 100 mM FeCl3 and equilibrated with 6% (v/v) acetic acid at pH 3.0 prior to sample loading. Tryptic peptides were dissolved in 6% (v/v) acetic acid and loaded onto the IMAC tip. Followed by 1% (v/v) trifluoroacetic acid, 80% ACN, and 6% (v/v) acetic acid washing steps, the bound phosphopeptides were eluted by 200 mM NH4H2PO4 and then desalted by SDB-XC StageTips47 and dried under vacuum. LC-SRM The enriched phosphopeptides or the unmodified peptide samples with spiked SI peptides were dissolved in 2% ACN/0.1% FA and analyzed using a TSQ Vantage triple quadruple mass spectrometer (Thermo Fisher Scientific) equipped with a nanoACQUITY UPLC system (Waters, Milford, MA). Peptide samples were loaded onto an ACQUITY UPLC BEH 1.7-µm C18 column (100 µm i.d. × 10 cm). The mobile phases were (A) 0.1% FA in water and (B) 0.1% FA in ACN. 2 µL of the unmodified peptide sample (0.3 µg) were loaded onto the column and separated at a flow rate of 400 nL/min using a 100-min gradient profile as follows (min:%B): 11:0.5, 13.5:0.5, 17:8, 25:13, 55:20, 80:38.5, 85:95, 89:50, 90:95, 91:0.5. For enriched phosphopeptide samples, all of the eluent from IMAC was dissolved in 10 µL and 4.5 µL were loaded onto the column and separated with the same LC gradient. The LC column is operated at a temperature of 44 ºC. The parameters of the triple quadruple instrument were set as follows: 0.7 fwhm Q1 resolution, and 1 s cycle time. Data were acquired in time-scheduled SRM mode (retention time window: 10 min). Data Analysis SRM data were analyzed using Skyline software. The total peak area ratios of endogenous light peptides and their heavy isotope-labeled internal standards (i.e., L/H peak area ratios) were calculated for quantification. Peak detection and integration were carried out based on two criteria: (1) same retention time and (2) similar relative SRM peak intensity ratios across multiple transitions between light (endogenous) peptides and the heavy SI peptide standards. All data were manually inspected to ensure correct peak detection and accurate integration.  

12 independent primary human breast tumor samples were used. Samples of human breast cancer have been described in CPTAC marker papers.