Provided by Emma (Yue) Zhang.
Peptide sequences were identified using Thermo Proteome Discoverer 1.3 from a human database SP.human.56.5 with full trypsin specificity and up to three internal missed cleavages. The tolerance was 50 ppm for precursor ions and 0.8 Da for product ions. Dynamic modifications were deamidation of asparagine, and static modification was carbamidomethylation for cysteine. Peptides were identified with Xcorr scores above the following thresholds: ≥3.8 for 3+ and higher charge state ions, ≥2.2 for 2+ ions, and ≥1.9 for 1+ ions.
Provided by Emma (Yue) Zhang.
Twenty microliters of lysis buffer (2% SDS in 50 mM NH4CO3) was added to 10 μL of cell lysate. Cells were solubilized by sonication using 20 s bursts, followed by cooling on ice for 20 s, in a process that was repeated for 10 times. The entire extract was concentrated down to 15 μL in a speed vacuum and loaded onto a gel (SDS-PAGE, 4−12% gradient) to separate proteins by molecular weight. After staining with Coomassie blue, each gel lane was cut into five individual slices as shown in Figure S1 (Supporting Information). Each slice was further minced into smaller pieces (approximately 0.5 mm2). The gel slices were washed with 600 mL of water for 15 min and centrifuged, supernatant was removed, and 50% ACN was added (1 mL), followed by shaking until no visible Coomassie stain remained. Proteins were then reduced with dithiothreitol (DTT) by adding 250 μL of 10 mMDTT in 0.1M NH4CO3 and incubated for 30 min at 56 °C. Samples were subsequently alkylated at room temperature and in the dark for 80 min with 250 μL of 55 mM iodoacetamide (IAA) in 0.1 M NH4CO3. Trypsin digestion reagent (200 μL; 10 ng/mL of trypsin in 50 mM NH4CO3, pH 8.0) was added, and samples were incubated for 30 min at 4 °C. The trypsin concentration was based upon an estimate of approximately 0.1−0.5 mg of protein per gel slice and adjusted as necessary. The solution was then replaced with 50 mM NH4CO3 to cover the gel pieces (50 μL) and incubated overnight at 37 °C to elute peptides from the gel. Following this step, supernatant was removed and stored. Gel pieces were further extracted with 5% formic acid (30 μL) and acetonitrile (ACN, 400 μL) at 37 °C for 10 min and then twice with 5% formic acid (30 μL) and ACN (200 μL). The formic acid solution containing tryptic peptides was combined with the previous supernatant and concentrated to 5−10 μL. The concentrated solution (trypsin-digested peptides) was subjected to LC−MS analysis.
Contact person William Hancock.
In view of the importance of EGFR/ERBB2 heterodimer signaling in breast cancer, it is of interest to explore the transcriptomic and proteomic analysis of two primary cell lines isolated from inflammatory breast cancer patients, one (SUM149) that expresses high levels of EGFR transcript with much lower levels of ERBB2 (1/4), while the other expresses very high levels of ERBB2 transcript (SUM190) and no detectable EGFR transcript. As a control we used a SKBR3 cell line that expressed high levels of ERBB2 transcript and low levels of EGFR. Analysis of the transcript levels indicated that the most likely signaling pathway for SUM190 involved the ERBB2/ERBB3 heterodimer, while SUM149 had several possibilities with involvement of EGFR dimers, ERBB2 heterodimers with EGFR and ERBB2 or ERBB3. We then explored the proteome of the two cell lines in terms of correlations between the transcriptome and proteomic measurements, identification of a panel of 21 oncogenes expressed in the two cell lines, interaction analysis of the observed proteins with this panel of oncogenes and selection of relevant cancer pathways. The analysis resulted in 4 pathways in addition to ERBB2 signaling (EGFR, integrin, MYC signaling, and PI3K signaling that contained many of the oncogene interacting proteins. In general there was reasonable agreement between the RNA-Seq and proteomic values shown in these tables except for some housekeeping proteins. In this study we have demonstrated that one of the goals of the chromosome-centric human proteome project (C-HPP), which is to integrate RNA-Seq with proteomics measurement.
The human breast cancer cell lines SKBR3 (ER/PR−, HER2+, metastatic pleural effusion), was obtained from the American Type Culture Collection (Manassas, VA) and maintained in culture with DMEM/F-12 medium supplemented with 10% FBS (Tissue Culture Biologicals, Seal Beach, CA) and 1% of Antibiotic-Antimycotic 100X (Gibco, Carlsbad, CA). SUM149 and SUM190 cells were obtained from Dr. Stephen Ethier (Kramanos Institute, MI, USA) and are commercially available (Asterand, Detroit, MI). SUM149 cells are ER/PR−, HER2− (triple receptors negative), and the SUM190 cells are ER/PR−, HER2+. Both human IBC cell lines were maintained in culture with Ham’s/F-12 medium supplemented with 10% FBS.
7. Data sets
RNA-Seq data sets, contac person Hogune Im
Strand-specific RNA-Seq libraries were prepared and sequenced on a lane of the Illumina HiSeq 2000 instrument per sample to obtain transcript data15. All RNAseq data are available at Short Read Archive (SRS366582, SRS366583, SRS366584, SRS366609, SRS366610, SRS366611).
We list here those.
proteins that were correlated with higher levels of EGFR or ERBB2 transcript, respectively.
EGFR signaling: caveolin 1 (CAV1), plectin (PLEC) (EGFR); growth factor receptor bound protein 7 (GRB7), Crk-like protein (CRKL) and Catenin delta-1 (CTNND1) (ERBB2).
Integrin signaling: filamin A (FLNA) and actinin alpha1 (ACTN1) (EGFR). MYC signaling: branched chain amino-acid transaminase 1 (BCAT1), carbamoyl-phosphate synthetase (CAD), nucleolin (NCL) (EGFR); transferrin receptor (TFRC), metadherin (MTDH) (ERBB2). p53 signaling: S100-A2 protein (S100A2), caveolin 1 (CAV1), Serpin B5 (SERPINB5), stratifin (SFN), PYD and CARD domain containing (PYCARD), and EPH receptor A2 (EPHA2) (EGFR).