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Dataset Information
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Dataset Name:
Breast Cancer Profiling Project  Proteomics 3: 1 total proteome dataset for a 27-cell line breast cancer panel under basal conditions 

Dataset Description:
We measured the baseline proteomic profiles of four non-malignant breast cell lines and 23 breast cancer cell lines of which three were triple negative, ten were hormone receptor positive, nine were Her2 amplified, and one was established from a triple negative patient-derived xenograft. 

--Data in Package:
HMS_Dataset_20370_DataFile.csv

--Metadata in Package:
20370_experimental_metadata.txt
Cell_Line_Metadata.txt

--Additional Information In Package:
BrCa_Proteomics3_readme.txt

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Center-specific Information
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Center-specific Name:
HMS_LINCS

Center-specific Dataset ID:
20370

Center-specific Dataset Link:
http://lincs.hms.harvard.edu/db/datasets/20370/

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Assay Information
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Assay Protocol:
1. Cells in mid-log phase of the growth cycle from 27 breast cancer cell lines were plated at appropriate densities in 15-cm plates to achieve ~50-60% confluence at the time of harvest. The growth conditions used are detailed in this file download: <a href="http://lincs.hms.harvard.edu/data/HMS_Dataset_20370_GrowthConditions.csv">Growth Conditions</a><br />
2. Cells were washed twice with ice cold PBS and scraped off plates in PBS containing 1X HALT protease/phosphatase inhibitor, pelleted, and stored at -80&deg;C until further processing.<br />
3. Working in batches of 9 or 10 samples, cell pellets were thawed, solubilized in lysis buffer (2% SDS, 150 mM NaCl, 50 mM Tris pH8.5, 1X HALT, 2 mM sodium vanadate), homogenized with a tissue homogenizer, reduced and alkylated. Based on BCA results, 4mg from each sample was subjected to methanol/chloroform precipitation.<br />
4. Precipitates were solubilized in 8M urea in 200 mM EPPS, diluted with further EPPS, and then underwent Lys-C and trypsin digests. A small aliquot was removed for a digestion check.<br />
5. After acidification with formic acid (FA), samples were purified via centrifugation, passage through low-protein binding filters, and C-18 Sep-Pak de-salting. From each sample, 60 &mu;g protein was aliquoted for proteomics, and the remainder of the sample used for phosphoproteomics.<br />
6. Samples were dried and resuspended in EPPS with acetonitrile (ACN) and labelled using tandem mass tag (TMT) 11plex peptide labelling reagents. A small aliquot was removed for a ratio check to ensure complete labeling (> 95%); equal amounts of labelled peptide from each sample (as judged from ratio check data) were combined for subsequent analysis. TMT labelling reactions were quenched by adding hydroxylamine to a final concentration of 0.5% (v/v).<br />
7. Samples were acidified, pooled, and de-salted prior to HPLC separation, using an Agilent 1200 Series instrument with a flow rate of 600&mu;l/min over a period of 75 minutes. Twelve fractions were collected over the last 65 minutes, and cleaned up using the Stage Tip protocol.<br />
8. Samples were dried, de-salted via stage tip, and resuspended in MS loading buffer (3% ACN, 5% FA), and injected into an Orbitrap Fusion Lumos Tribrid MS using a multi-notch MS3 method. LC-MS was performed in the Orbitrap over a scan range of 400-1400m/z with dynamic exclusion. Turbo rate scans were performed in the Ion Trap with a collision energy of 35% and a maximum injection time of 200 ms. TMT quantification was performed using SPS-MS3 in the Orbitrap with a scan range of 100-1000 m/z and an HCD collision energy of 55%.<br />
9. A compilation of commercially available software (Core software program) was used to convert mass spectrometric data (Thermo .RAW files) to mzXML format and correct monoisotopic m/z measurements and erroneous peptide charge state assignments. Assignment of MS/MS spectra was performed using Sequest and the Human Uniprot database. 

Date Updated:
2020-03-02

Date Retrieved from Center:
2020-6-12

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Metadata Information
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Metadata information regarding the entities used in the experiments is included in the accompanied metadata. A metadata file per entity category is included in the package. For example, the metadata for all the cell lines that were used in the dataset are included in the Cell_Lines_Metadata.txt file.
Descriptions for each metadata field can be found here: http://www.lincsproject.org/data/data-standards/
