Large-Scale Analysis of Breast Cancer-Related. Conformational Changes in Proteins using SILAC-SPROX. *Corresponding author

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1 SUPPORTING INFORMATION for: Large-Scale Analysis of Breast Cancer-Related Conformational Changes in Proteins using SILAC-SPROX Fang Liu, 1 He Meng, 1 and Michael C. Fitzgerald 1, * 1 Department of Chemistry, Duke University, Durham, North Carolina *Corresponding author The Supporting Information includes: Supplementary Text. Including the cell culture procedure and a summary of the constraints used in the regression analysis for hit identification. Supplementary Table S-1. Excel spreadsheet summarizing methioninecontaining peptides assayed in the three biological replicates of MCF-7 versus BT-474 cell line comparison. Supplementary Table S-2. Excel spreadsheet summarizing methioninecontaining peptides assayed in the three biological replicates of MCF-7 versus MDA-MB-468 cell line comparison. Supplementary Table S-3. Excel spreadsheet summarizing peptide and protein hits identified with altered thermodynamic stabilities in the MCF-7 versus BT-474 cell line comparison. Supplementary Table S-4. Excel spreadsheet summarizing peptide and protein hits identified with altered thermodynamic stabilities in the MCF-7 versus MDA- MB-468 cell line comparison. S-1

2 Supplementary Table S-5. Excel spreadsheets summarizing relative protein expression levels in the MCF-7 versus BT-474 cell line comparison. Supplementary Table S-6. Excel spreadsheets summarizing relative protein expression levels in the MCF-7 versus MDA-MB-468 cell line comparison. Supplementary Figure S-1. Fitted SILAC-SPROX curves for all the peptide hits identified with altered thermodynamic stabilities in the MCF-7 versus BT-474 cell line comparison. Supplementary Figure S-2. Fitted SILAC-SPROX curves for all the peptide hits identified with altered thermodynamic stabilities in the MCF-7 versus MDA-MB- 468 cell line comparison. Supplementary Figure S-3. Global distributions of the H/L ratios determined for all the non-methionine-containing peptides identified in the (A) MCF-7 versus BT- 474 and (B) MCF-7 versus MDA-MB-468 cell line comparisons. S-2

3 SUPPLEMENTARY TEXT Cell Culture Procedure The cell culture models of breast cancer used in this work included the MCF-7, BT-474 and MDA-MB-468 cell lines. They were acquired from the American Type Culture Collection (provided by the Cell Culture Facility at Duke University). The BT-474 cells were maintained in RPMI 1640 medium (Sigma) containing 2.5 g/l glucose (Sigma), 10 mm HEPES (Gibco), 1 mm sodium pyruvate (Gibco), 10% fetal bovine serum (FBS) (Hyclone) supplemented with 10 μ g/ml of insulin (Gibco). The MDA-MB-468 cells were maintained in Leibovitz s L-15 medium (Gibco) containing 10% FBS. The MCF-7 cells were initially propagated in DMEM medium (Gibco) containing 1 mm sodium pyruvate, 0.1 mm non-essential amino acids (NEAA) (Gibco) and 10% FBS supplemented with 10 μg/ml of insulin. The MCF-7 cells were passaged twice in this medium and then transferred to DMEM medium containing all of the above except 10% dialyzed FBS was used instead of the regular serum. The cells were adapted well in this medium before passaging on the heavy SILAC medium. The heavy SILAC medium comprised of the SILAC DMEM (Thermo Scientific), 10% dialyzed FBS (Sigma), 10 μg/ml of insulin with 500 μl of 13 C 6 Arginine (86.2 mg/l) and 500 μl of 13 C 15 6 N 2 Lysine (181.2 mg/l) (Cambridge Isotope Laboratories) and 500 μl of Proline (200mg/L) (Sigma). The MCF-7 cells were passaged at least four times in the heavy SILAC medium before they were harvested. All cells were maintained in a humidified 37 incubator with 5% CO 2 except the MDA-MB-468 cells were cultured in the absence of CO 2. All cells were washed twice with PBS before they were S-3

4 harvested. The BT-474 and MDA-MB-468 cells were harvested with 0.25% (w/v) Trypsin/0.53 mm EDTA solution (Gibco). The MCF-7 cells were harvested with HyQtase solution (Hyclone). The harvested cells were pelleted at 1000 rpm for 5 min. The resulting cell pellets were washed with PBS and stored at -80. Constraints Used in the Regression Analysis for Hit Identification Global Fitting Constraints: 0.01 < A < 1 20 < G < < m. < < m 0 < < m. m 0 < 2 Other Fitting Constraints: Concavity or convexity was determined by the point with largest log 0 H/L, denoted by [D] : and [log 0 H/L ] :. If [log 0 H/L ] : > 0, then 20 < G < 0.01 If [log 0 H/L ] : < 0, then 0.01 < G < 20 [D] : 0.5 C./0,?@ABCDEAB < 2 G < [D] m. + m : C./0,?@ABCDEAB 0, where C./0,?@ABCDEAB is the width of maximum consecutive points peak. S-4

5 (A) (B) Figure S-3. Global distributions of the log 2 (normalized H/L) values determined for all the non-methionine-containing peptides identified at all the denaturant concentrations in the (A) MCF-7 versus BT-474 and (B) MCF-7 versus MDA- MB-468 cell line comparisons. The arrows indicate the 5 th and 95 th percentile cut-off values in each biological replicate. The median log 2 (normalized H/L) values were 0 in all distributions and the standard deviations ranged from S-5