Supporting Information Supporting Information Corrected July 15 , 2014 Yi et al. 10.1073/pnas.1404943111 SI Materials and Methods which no charge state could be determined were excluded MS/ Tumorsphere Growth Assay. Single-cell tumorsphere growth assays MS selection). Three independent experiments were performed. of human mammary epithelial (HMLER) (CD44high/CD24low)SA and HMLER (CD44high/CD24low)FA population cells were per- Phosphorylation Site Localization. The probability of correct phos- formed with ultra-low attachment surface six-well plates (Corn- phorylation site localization for each phosphorylation site was ing) and the mammary epithelial cell basal medium (MEBM) measured using an Ascore algorithm (1). This algorithm con- medium (Lonza) was supplemented with B27 (Invitrogen), 20 ng/mL siders all phosphoforms of a peptide and uses the presence or EGF, and 20 ng/mL basic FGF (BD Biosciences), and 4 μg/mL absence of experimental fragment ions unique to each to create an ambiguity score (Ascore). Parameters included a window size of heparin (Sigma). Single cells were cultured for 8 d and images were ± taken with a Nikon camera. 100 m/z units and a fragment ion tolerance of 0.6 m/z units. Sites with an Ascore of ≥13 (P ≤ 0.05) were considered to be ≥ ≤ Soft Agar Colony Growth Assay. Single-cell soft agar colony for- confidently localized, and those with an Ascore of 19 (P 0.01) mation and growth assays were performed to identify the tumor- were considered to be localized with near certainty. More than ≥ igenesis capacity of HMLER (CD44high/CD24low)SA and HMLER a 2.5-fold ratio ( 60.02%) was considered as a significant (CD44high/CD24low)FA population cells in vitro. Briefly, base agar phosphorylation increase in phosphoproteome assays. As cellu- layer was 0.5% agar with MEBM, the top agar layer was 0.35% agar lar active kinases are rapidly dephosphorylated by phospha- ∼ with mammary epithelial cell growth medium (MEGM) (500 mL tases in cells (2, 3), 24 kinases within 1.6- to 2-fold ratio (an ∼ MEBMwith2mLBPE,0.5mLinsulin,0.5mLrhEGF,0.5mL increase from 37.5% to 50%) were tracked and recovered as GA-1000, 0.5 mL hydrocortisone; Lonza). HMLER (CD44high/ phosphorylation increased kinases. A decrease of more than CD24low)SA and HMLER (CD44high/CD24low)FA population cells 60% was considered a significant phosphorylation decrease. (1 × 104) in single-cell suspensions were mixed with 2 mL 0.35% The postsearch data analyses were performed as previously de- agar with MEGM, and then the mixture was added onto the base scribed (4). agar layer of each well in the six-well plates. After about 2 wk, Database Searches, Data Filtering, Validation of Protein Detection grossly visible colonies appeared. Images were taken with a Nikon Rate, and Phosphoproteome Analyses. The spectral data were camera. Colony diameters were analyzed and numbers counted searched with SEQUEST (1) against a database containing the with ImageJ software (http://imagej.nih.gov/ij/). human protein sequence database (www.ensembl.org/index.html) high low SA together with the reversed complement. The LC-MS/MS identi- Cell Viability Assay. HMLER (CD44 /CD24 ) and HMLER (CD44high/CD24low)FA population cells (1 × 104) were treated fications were filtered to 0.98% protein false discovery rate (FDR) and 0.1% peptide FDR. The peptide quantification and with indicated compounds with series of concentrations for 24 h. phosphorylation site localization were analyzed using in-house Cell viability assays were performed on the cells with CellTiter- software and Ascore as previously described (5). Glo luminescent cell viability assay kit (Promega) according to the manual. Three independent experiments were performed. Peptide Quantification. Peptide quantification was performed us- ing the Vista program. We required a signal-to-noise ratio (S/N) Strong Cation Exchange/Immobilized Metal Affinity Chromatography value >3 for both heavy and light species for quantification. For Phosphopeptide Enrichment and Liquid Chromatography Tandem peptides found exclusively as singlets (only a heavy or only a light Mass Spectrometry Analyses. In phosphopeptide enrichment by peak present), we reported the peak S/N ratio or its inverse, as strong cation exchange/immobilized metal affinity chromatog- a proxy for relative abundance measurement. For such peptides, raphy (SCX/IMAC), peptides were first separated at acidic pH on we required an S/N value >5 for the observed species. In addi- a polysulfethyl A semipreparative column (9.4 mm i.d. × 200 mm tion, if the S/N value of one member of a pair was <3, the length, 5-μm particle size, 200-Å pore size; PolyLC). Twelve partner value was required to be >5. Finally, to avoid quantifying fractions were collected and further enriched by IMAC, starting false positives, any identification from a singlet peak was re- with 30 μL of PHOS-Select beads (Sigma-Aldrich). Enriched quired to pass an identification threshold that was 10 times more phosphopeptides were eluted and desalted by STAGE-TIPS stringent (Q value of <0.001; precision >99.9%). Raw abun- (Thermo Scientific) as previously described (1). Phosphopep- dance ratios from each experiment were normalized based on tide-enriched fractions were analyzed by liquid chromatography the median distribution ratio. tandem mass spectrometry (LC-MS/MS) on an LTQ Orbitrap Velos mass spectrometer (Thermo Scientific) equipped with Accessibility of Raw Data and Bioinformatic Analysis. The five Excel a Thermo Fisher Scientific nanospray source, an Agilent 1100 files (Files S1–S5) and MS raw data are available at http:// Series binary HPLC pump, and a Famos autosampler. Phos- gwagner.med.harvard.edu/intranet/PNAS_Manuscript_2014/. phopeptides were separated on a 0.125 × 180 mm fused silica microcapillary column with an in needle tip (made in-house) Western Blot Assay. Total proteins were extracted from breast with a ∼5-μm i.d. The silica microcapillary column was packed cancer stem cells (CSCs) with protease inhibitor (Roche) and with magicC18AQ C18 reverse-phase resin (5-μm particle size, phosphatase inhibitor (Sigma) for Western blot assays with 200-Å pore size; Michrom Bioresources). Separation was per- specific antibodies. Anti-p120 catenin and anti–p-p120 (Tyr280) formed by applying a 57-min gradient from 7% to 28% aceto- catenin antibodies were obtained from Syd Labs. Anti-GSK3β, nitrile in 0.125% formic acid. The mass spectrometer was anti-p(Ser9) GSK3β, anti-PKAC, anti-p(Thr197) PKAC, anti- operated with default settings: full MS [automatic gain control ERK1/2, anti-p(Thr202/Tyr204)-ERK1/2, anti-p(Ser217/221) (AGC), 1 × 106; resolution, 6 × 104; m/z range, 375–1,800; MEK1/2, anti-MEK1/2, anti-chemokine (C-X-C motif) re- maximum ion time, 1,000 ms]; MS/MS (AGC, 5 × 103; maximum ceptor 4 (CXCR4), anti–p-Ser/Thr, anti–β-actin, and anti-tubulin ion time, 120 ms; minimum signal threshold, 4 × 103; dynamic antibodies were ordered from Cell Signaling. Anti–p-Tyr (mono- exclusion time setting, 30 s; singly charged ions and ions for clonal) antibodies were ordered from Abcam. PKA inhibitor Yi et al. www.pnas.org/cgi/content/short/1404943111 1of15 (14-22-Amide) was ordered from EMD. AMD3100 (A5602) was (http://networkin.info/search.php), and Kinasource (www.kinasource. ordered from Sigma. The short hairpin RNA (shRNA)-CXCR4 co.uk/Database/substrates.html) (9), phosphotase–substrates with plasmids and control shRNA plasmids were ordered from The PubMed, and pathways with the Kyoto Encyclopedia of Genes RNAi Consortium (Broad Institute). Active SDF-1 protein was and Genomes (www.genome.jp/kegg/pathway.html). The kinase– obtained from Abcam (ab78808). Each experiment was repeated phosphorylation site-specific substrate relationships were either at least three times. supplied in Kinasource or reported in the literature (Table S2, Cellular protein extraction and Western blot analysis was S3, and S7). All relationships of the kinase–substrate and performed with radioimmunoprecipitation assay buffer (50 mM phosphotase–substrate in this study are experimentally identified Tris·HCl pH 7.4, 150 mM NaCl, 1% Triton-X100, 0.1% SDS, × in human cells in previously published reports (refs. 5, 9, 10, and 0.25% Na-deoxycholate, 1 mM PMSF, 1 Roche complete mini 11 and Tables S3 and S7). The results were compared with 40 protease inhibitor mixture, 1× Pierce phosphatase inhibitor phosphoproteomic experiments of mammalian cells in the lit- mixture). The total Ser/Thr, or Tyr phosphorylation signal in- erature to validate the prediction for the biological function, tensities in Western blots were analyzed by Bio-Rad imaging system analysis software. The ratios of phosphorylation signal activation, and/or inhibition of phosphorylation sites. The net- intensities of samples compared with the control were expressed works were seeded on established SDF-1/CXCR4 signaling – as relative phosphorylation activities. pathways (12 14) with extended signal transduction based on the above-mentioned integrated bioinformatic analyses of phospho- Bioinformatic Analysis. Bioinformatic annotations were carried out proteins in the phosphoproteome. using Human Protein Reference Database (www.hprd.org)as previously described (4, 6). The phosphosites were identified Conserved Phosphorylation Site Analysis Across Species. Conserved using the Web-based program www.phosphosite.org and phosphorylation sites and motifs were analyzed with target PubMed database (www.ncbi.nlm.nih.gov/pubmed). The pre- protein sequences in Homo sapiens (PubMed protein database, viously detected phosphosites with unknown biological function www.ncbi.nlm.nih.gov/protein)andBLAST(http://blast.ncbi.nlm. are not labeled. LC-MS/MS data were analyzed for kinase– nih.gov/Blast.cgi) search of the following species: Homo sapiens, substrates using NetPhorest (7) (http://netphorest.info), for Mus musculus, Drosophila melanogaster, Caenorhabditis elegans, kinase–substrate network reconstruction using NetworKIN (8) Arabidopsis thaliana, and Saccharomyces cerevisiae. 1. Villén J, Gygi SP (2008) The SCX/IMAC enrichment approach for global phosphorylation 9. Hernandez M, Lachmann A, Zhao S, Xiao K, Ma’ayan A (2010) Inferring the sign of analysis by mass spectrometry.