Quantitative Proteomics Analysis of Breast Cancer

RIME^{22 (link)} and stable isotope dimethyl labeling^{21 (link)} were performed, as previously described. The wt-SUM44 and SUM44-LTED were labeled with the medium and light isotope reagent, respectively. Labeled samples were pooled at an approximate 1:1 ratio, dried down and fractionated using 12 cm IPG strip pH 3–10, as previously described^{46 (link)}. RIME and dimethyl label fractions were desalted (SUM SS18V, The Nest Group Inc) and run through LC-MS/MS using LTQ Velos Orbitrap MS. The data acquisition mode was set, as previously described^{46 (link)}. Raw data for RIME and dimethyl labeling were analyzed using MaxQuant 1.5.1.0^{46 (link),47 (link)}. Search parameters were as previously described^{46 (link)}. All proteomics data are deposited within the PRIDE database (PXD004807).

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Martin L.A., Ribas R., Simigdala N., Schuster E., Pancholi S., Tenev T., Gellert P., Buluwela L., Harrod A., Thornhill A., Nikitorowicz-Buniak J., Bhamra A., Turgeon M.O., Poulogiannis G., Gao Q., Martins V., Hills M., Garcia-Murillas I., Fribbens C., Patani N., Li Z., Sikora M.J., Turner N., Zwart W., Oesterreich S., Carroll J., Ali S, & Dowsett M. (2017). Discovery of naturally occurring ESR1 mutations in breast cancer cell lines modelling endocrine resistance. Nature Communications, 8, 1865.

Publication 2017

SUM SS18V

Isotope Light Ms ms

Corresponding Organization : Institute of Cancer Research

Other organizations : Imperial College London, University of London, Royal Marsden Hospital, University of Pittsburgh, The Netherlands Cancer Institute, Oncode Institute, Cancer Research UK, University of Cambridge

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Variable analysis

independent variables

Labeling of wt-SUM44 and SUM44-LTED with medium and light isotope reagent, respectively

dependent variables

Proteomics data obtained from RIME and dimethyl labeling experiments

control variables

Fractionation of labeled samples using 12 cm IPG strip pH 3–10
Desalting of RIME and dimethyl label fractions using SUM SS18V (The Nest Group Inc)
LC-MS/MS analysis using LTQ Velos Orbitrap MS
Data acquisition mode as previously described

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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