Robust Quantification of Breast Cancer Proteins

RPPA was completed independently and at different time points for training and tests sets using individual arrays. Protein was extracted from human tumors and RPPA was performed as described previously [16 -19 (link)]. Lysis buffer was used to lyse frozen tumors by homogenization (excised tumors) or sonication (FNAs). Tumor lysates were normalized to 1 μg/μL concentration as assessed by bicinchoninic acid assay (BCA) and boiled with 1% SDS. Supernatants were manually diluted in five-fold serial dilutions with lysis buffer. An Aushon Biosystems 2470 arrayer (Burlington, MA) created 1,056 sample arrays on nitrocellulose-coated FAST slides (Schleicher & Schuell BioScience, Inc.). Slides were probed with 146 validated primary antibodies (Additional File 1, Table S1) and signal amplified using a DakoCytomation-catalyzed system. Secondary antibodies were used as a starting point for amplification. Slides were scanned, analyzed, and quantified using Microvigene software (VigeneTech Inc., Carlisle, MA) to generate spot signal intensities, which were processed by the R package SuperCurve (version 1.01) [18 (link)], available at "http://bioinformatics.mdanderson.org/OOMPA". A fitted curve ("supercurve") was plotted with the signal intensities on the Y-axis and the relative log2 concentration of each protein on the X-axis using the non-parametric, monotone increasing B-spline model [18 (link)]. Protein concentrations were derived from the supercurve for each lysate by curve-fitting and normalized by median polish. Protein measurements were corrected for loading as described [15 (link)-17 (link),19 (link)]. For the selection of the 146 antibody set, we focused on markers currently used for breast cancer classification due to their value in treatment decisions (ER, PR, HER2). We then added additional antibodies to targets implicated in breast cancer pathophysiology, followed by antibodies to targets implicated in the pathophysiology of other cancer lineages. Final selection of antibodies was also driven by the availability of their high quality that could pass a strict validation process as previously described [20 (link)].

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Gonzalez-Angulo A.M., Hennessy B.T., Meric-Bernstam F., Sahin A., Liu W., Ju Z., Carey M.S., Myhre S., Speers C., Deng L., Broaddus R., Lluch A., Aparicio S., Brown P., Pusztai L., Symmans W.F., Alsner J., Overgaard J., Borresen-Dale A.L., Hortobagyi G.N., Coombes K.R, & Mills G.B. (2011). Functional proteomics can define prognosis and predict pathologic complete response in patients with breast cancer. Clinical proteomics, 8(1), 11.

Publication 2011

Antibodies Antibody Assay Axis Bicinchoninic acid Breast cancer Buffer Cancer Dilutions Fnas Frozen Her2 Human Nitrocellulose Protein Tumor

Corresponding Organization : The University of Texas MD Anderson Cancer Center

Other organizations : Norwegian Cancer Society, University of Oslo, Baylor College of Medicine, Hospital Clínico Universitario de Valencia, University of British Columbia, Aarhus University Hospital

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Protocol cited in 21 other protocols

Variable analysis

independent variables

Type of tumor samples used (excised tumors or FNAs)
Lysis buffer used to lyse the tumor samples (homogenization or sonication)

dependent variables

Protein concentrations derived from the supercurve for each lysate by curve-fitting and normalized by median polish

control variables

Tumor lysates were normalized to 1 μg/μL concentration as assessed by bicinchoninic acid assay (BCA)
Tumor lysates were boiled with 1% SDS
Tumor lysates were manually diluted in five-fold serial dilutions with lysis buffer
An Aushon Biosystems 2470 arrayer created 1,056 sample arrays on nitrocellulose-coated FAST slides
Slides were probed with 146 validated primary antibodies
Signal was amplified using a DakoCytomation-catalyzed system
Secondary antibodies were used as a starting point for amplification
Slides were scanned, analyzed, and quantified using Microvigene software
Protein measurements were corrected for loading as described in references [15-17, 19]

controls

Positive control: No positive control is explicitly mentioned.
Negative control: No negative control is explicitly mentioned.

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