Breast Cancer Profiling Project – Proteomics 3: 1 total proteome dataset for a 27-cell line breast cancer panel under basal conditions - Dataset (ID:20370)
|HMS Dataset ID:||20370|
|Dataset Title:||Breast Cancer Profiling Project – Proteomics 3: 1 total proteome dataset for a 27-cell line breast cancer panel under basal conditions|
|Screening Lab Investigator:||Kartik Subramanian, Caitlin E. Mills, Maulik Nariya, Marian Kalocsay, Matt Berberich|
|Screening Principal Investigator:||Peter K. Sorger|
|Assay 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.|
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: Growth Conditions|
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°C until further processing.
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.
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.
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 μg protein was aliquoted for proteomics, and the remainder of the sample used for phosphoproteomics.
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).
7. Samples were acidified, pooled, and de-salted prior to HPLC separation, using an Agilent 1200 Series instrument with a flow rate of 600μl/min over a period of 75 minutes. Twelve fractions were collected over the last 65 minutes, and cleaned up using the Stage Tip protocol.
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%.
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.
|Assay Protocol Reference:||
Rappsilber, J.; Mann, M.; Ishihama, Y., Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips. Nature Protocols 2007, 2 (8), 1896-1906|
Ting, L.; Rad, R.; Gygi, S. P.; Haas, W., MS3 eliminates ratio distortion in isobaric multiplexed quantitative proteomics. Nature Methods 2011, 8 (11), 937-940
McAlister, G. C.; Nusinow, D. P.; Jedrychowski, M. P.; Wuhr, M.; Huttlin, E. L.; Erickson, B. K.; Rad, R.; Haas, W.; Gygi, S. P., MultiNotch MS3 enables accurate, sensitive, and multiplexed detection of differential expression across cancer cell line proteomes. Anal Chem 2014, 86 (14), 7150-8
Eng, J. K.; McCormack, A. L.; Yates, J. R., An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database. Journal of the American Society for Mass Spectrometry 1994, 5 (11), 976-989
|HMS Dataset Type:||Proteomics|
|Date Publicly Available:||2020-03-02|
|Most Recent Update:||2020-03-02|