Recombinant human p53 (rp53) samples will be isolated from an E. coli expression system by cobalt affinity chromatography, modified in vitro, processed directly or subjected to proteolytic digest, desalted, and analyzed by top-down (TD), middle-down (MD), or bottom-up (BU) proteomics on a QE-HF or Orbitrap Fusion Lumos mass spectrometer (MS, both Thermo Fisher Scientific) at Northwestern University (NU). Additional samples will be subjected to proteolytic digest and analyzed by multiple reaction monitoring (MRM) analysis on a TSQ Quantiva MS (Thermo Fisher Scientific) at NU. Endogenous p53 (ep53) samples will be produced at and provided to NU by the Lahav laboratory at Harvard Medical School (HMS). If needed, additional ep53 samples will be produced at and processed directly by NU. All ep53 samples will be isolated from MCF7 whole-cell lysates by immunoprecipitation (IP), processed directly or subjected to proteolytic digest, desalted, and analyzed by TD, MD, or BU proteomics on a QE-HF or Orbitrap Fusion Lumos MS at NU. Additional samples will be subjected to proteolytic digest and analyzed by MRM analysis on a TSQ Quantiva MS at NU. If needed, NU will produce and ship rp53 and ep53 samples to the National High Magnetic Field Laboratory (Tallahassee, FL) for analysis by TD and MD proteomics on the 21 Tesla FT-ICR mass spectrometer, in collaboration with Lissa Anderson and Christopher Hendrickson. All TD, MD, BU, and MRM data analysis will be performed by NU. Data interpretation and integration will be performed in collaboration with Jeremy Gunawardena and Galit Lahav of HMS.
|Effective start/end date||9/15/19 → 6/30/23|
- Harvard University (153447.5111019.0005//2R01GM105375-05)
- National Institute of General Medical Sciences (153447.5111019.0005//2R01GM105375-05)