Quantitative data for differentially expressed phosphopeptides in MCF7 cells treated with palbociclib or abemaciclib at either 0.3 or 3.0 μM and untreated controls - Dataset (ID:20371)

HMS Dataset ID: 20371
Dataset Title: Quantitative data for differentially expressed phosphopeptides in MCF7 cells treated with palbociclib or abemaciclib at either 0.3 or 3.0 μM and untreated controls
Project Summary Page(s): Publication Summary
Screening Lab Investigator: Caitlin Mills
Screening Principal Investigator: Peter K. Sorger
Assay Description: Total phosphoproteomics of MCF7 cells treated with Abemaciclib, Palbociclib (0.3 or 3 µM), or DMSO control for one hour. Log2 fold change data are provided.
Assay Protocol: 1. 5 x 15 cm plates were seeded with 2.5 x 10^6 MCF7 cells per condition in duplicate. Three days after plating, cells were treated with 10 ml spent media containing drug treatment (Palbociclib or Abemaciclib at 0.3 µM or 3 µM) or DMSO control for one hour and then harvested.
2. Cells were washed twice with ice cold PBS and scraped off of the plates in PBS containing 1X HALT protease/phosphatase inhibitor, pelleted, and stored at -80°C until further processing.
3. Working in batches of 9 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 handheld drill, reduced and alkylated. Based on BCA results, 4.5 mg from each sample was subjected to methanol/chloroform precipitation.
4. Precipitates were solubilized in 8M urea in 200 mM further diluted with EPPS, and then underwent Lys-C and trypsin digests. A small aliquot was removed for a digestion check (< 15% missed cleavages).
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.
6. The samples were dried and enriched for phosphopeptides using a Thermo Scientific High-Select Fe-NTA Phosphopeptide Enrichment Kit according to the manufacturer's directions. An aliquot was removed to monitor phosphopeptide enrichment efficiency.
7. Samples were labelled using a TMT10plex Mass Tag Labelling Kit. A small aliquot was removed for a ratio check to ensure complete labeling (> 98%); 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).
8. Phosphotyrosine-containing peptides were enriched by incubation at 4°C with p-Tyr-1000 rabbit antibody coupled to protein A-agarose beads. Phosphotyrosine peptides were eluted twice with 0.15% FA, followed by Stage Tip desalting.
9. HPLC fractionation was then performed on the phosphotyrosine-IP flow-through using an Agilent 1200 Series instrument with a flow rate of 600μl/min over a period of 75 minutes. Fractions were collected over the last 65 minutes and pooled into 24 samples, followed by sample cleanup using the Stage Tip protocol.
10. Samples were dried 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. The top 10 ions with charge states from 2 to 6 were selected for MS/MS. Rapid rate scans were performed in the Ion Trap with a collision energy of 35% and a maximum injection time of 120 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%.
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 (v28) and the Human Uniprot database (downloaded 2/4/2014).
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: Phosphoproteomics
Date Publicly Available: 2020-06-24
Most Recent Update: 2020-06-24