Specificity of phosphorylation responses to MAP kinase pathway inhibitors in melanoma cells, experiment 1 of 2
We used phosphoproteomics to compare the responses of the ERK1/2 inhibitors, SCH772984 and GDC0994, and the MKK1/2 inhibitor, trametinib. These are compared with responses to the MKK1/2 inhibitor, selumetinib (AZD6244), previously measured by our lab in the same metastatic melanoma cell line. In three replicate experiments, we quantified a total of 12,805 class I phosphosites on 3,819 proteins in the trametinib-SCH772984-DMSO experiment, and 7,074 class I phosphosites on 2,453 in the GDC0994-SCH772984-DMSO experiment. This included 466 phosphosites that reproducibly decreased in response to at least one inhibitor in the trametinib-SCH772984-DMSO experiment and 414 phosphosites in the GDC0994-SCH772984-DMSO experiment. The results demonstrate linearity in signaling through the MAP kinase pathway. By comparing multiple inhibitors targeted to multiple tiers of protein kinases in the MAPK pathway, we gain insight into regulation and new targets of the oncogenic BRAF driver pathway in cancer cells, and a useful approach for evaluating the specificity of drugs and drug candidates. SILAC Experimental Design Experiment 1 Replicate 1: Heavy – DMSO, Medium – SCH772984, Light – Trametinib Replicate 2: Heavy – SCH772984, Medium – Trametinib, Light – DMSO Replicate 3: Heavy – Trametinib, Medium – DMSO, Light – SCH772984 SILAC Experimental Design Experiment 2 Replicate 1: Heavy – DMSO, Medium – SCH772984, Light – GDC0994 Replicate 2: Heavy – SCH772984, Medium – GDC0994, Light – DMSO Replicate 3: Heavy – GDC0994, Medium – DMSO, Light – SCH772984 File List 1. Zipped MaxQuant search results folder containing index and output folders for each raw file, ‘combined’ output folder, and mqpar.xml MaxQuant search parameters file 2. Individual raw files of phosphopeptide-enriched ERLIC fractions and total protein fractions 3. Zipped MaxQuant version used for analysis 4. FASTA file containing Uniprot human identifications 5. Instructions for viewing annotated spectra
Sample Processing Protocol
WM239A cells were SILAC-labeled using SILAC RPMI media supplemented with heavy, medium, or light isotopically-labeled arginine (40 µg/mL) and lysine (200 µg/mL), 180 mg/L proline, 10% (v/v) dialyzed FBS, penicillin (100 µg/mL), and streptomycin (100 µg/mL). Cells were incubated in a humidified chamber maintained at 37°C with 5% CO2. WM239A cells were grown in heavy (H), medium (M) or light (L) SILAC RPMI, and plated overnight at 35E6 cells/dish. The following day, each dish was treated with inhibitor (10 µM) or DMSO for 2 hrs. Lysates from each heavy, medium, and light dish were then mixed and phosphopeptide samples were prepared as previously described in Stuart SA, et al. Mol Cell Proteomics. 2015 Jun:14(6):1599-615. Proteomes were sampled prior to phosphoenrichment and high pH reverse-phase fractionated using an M-class Acquity UPLC, with an in-house prepared C18 column (1.8 µm 120Å UChrom, nanolcms). Tryptic peptides were loaded onto the column equilibrated with 5% acetonitrile, 10mM ammonium formate, pH 10, and eluted with a 170 min linear gradient to 80% acetonitrile, 10mM ammonium formate, pH 10. Fractions were collected in a concatenated fashion throughout the gradient for a total of twelve fractions. For SILAC experiments comparing DMSO-trametinib-SCH772984, LC-MS/MS analysis was performed with an Orbitrap Fusion with either a nanoLC1000 or an M-class Acquity UPLC. Phospho-enriched peptides were loaded directly onto an M-class Acquity Peptide BEH C18 column and eluted with a gradient from 3-20% acetonitrile in 100 min, 20-32% acetonitrile in 20 min, and 32-85% acetonitrile in 1 min holding 85% for 4 min including 0.1% formic acid. MS1 scans were collected at 120,000 resolution from 380-1500 m/z with an AGC target of 2E5 and a max inject fill time of 50 ms. MS2 scans were acquired in top speed mode for 3 sec on the most intense ions with a quadrupole isolation window of 1.6 Da and normalized collision energy (NCE) of 35% for HCD fragmentation. Fragment ions were collected in either the Orbitrap or the linear ion trap. Total protein samples were analyzed by LC-MS/MS using the same method. For the DMSO-SCH772984-GDC0994 SILAC experiments, LC-MS/MS analysis was performed using Waters Acquity UPLC and LTQ Orbitrap Velos system with gradient elution and instrument settings as previously described in Stuart SA, et al. Mol Cell Proteomics. 2015 Jun:14(6):1599-615.
Data Processing Protocol
Raw MS files for phosphopeptide and total peptide fractions were analyzed together using the Andromeda search engine in MaxQuant (v126.96.36.199) software and processed with Perseus software (v188.8.131.52) using default settings as previously described in Stuart SA, et al. Mol Cell Proteomics. 2015 Jun:14(6):1599-615. MaxQuant identifies common contaminants and peptides matching to a target-decoy database containing reversed versions of each peptide in the uploaded Uniprot databse. The previously published DMSO-selumetinib-vemurafenib dataset (Stuart SA, et al. Mol Cell Proteomics. 2015 Jun:14(6):1599-615) was also reprocessed with these software versions. Searches used the Uniprot human proteome reference (08-21-2015 download, with 21,051 entries). Mass tolerances were 20 ppm for FTMS precursor ions and 0.5 Da for ITMS MS/MS ions. The minimum peptide length was 7 amino acids. MaxQuant default score cutoffs of 0 for unmodified peptides and 40 for modified peptides were used. False discovery rates were 1% for both phosphopeptide and protein identifications. For protein quantification, the minimum number of total peptides was 2 (unique + razor). Phosphorylated peptides and their unmodified counterparts were excluded for protein quantification. Raw files for phosphopeptide data and total protein data were defined as separate groups. Multiplicity was set to 3 with medium labels of Arg6 and Lys4 and heavy labels of Arg10 and Lys8 for both groups. For phosphopeptide files, Phospho(STY) was set as a variable modification. All files were searched with carbamidomethylation (Cys) as a fixed modification and acetylation (N-term) and oxidation (Met) as variable modifications. Localization probabilities were assessed with the MaxQuant PTM score. The enzyme specificity was trypsin/P and 2 missed cleavages were allowed. For phosphopeptide data, the Phospho(STY) output file was uploaded into Perseus. Reverse and contaminant rows were removed and the site table was expanded so that ratios for singly, doubly, and “triply” (3 or more phosphates) phosphorylated peptides were treated as individual phosphosites. Rows not quantified in any of the experiments after expanding the site table were removed. For total peptide samples, the protein groups output file was uploaded into the Perseus and rows not quantified or designated as reverse, contaminant, and only identified by site were removed.
Basken J, Stuart SA, Kavran AJ, Lee T, Ebmeier CC, Old W, Ahn NG. Specificity of phosphorylation responses to MAP kinase pathway inhibitors in melanoma cells. Mol Cell Proteomics. 2017 PubMed: 29255136