MATRIX platform to analyze translation machinery remodeling in glioblastoma cells

  • STAR Protoc. 2022 Dec 16;3(4):101919. doi: 10.1016/j.xpro.2022.101919.
J J David Ho  1 ,  Tyler A Cunningham  2 ,  Jonathan R Krieger  3 ,  Jonathan H Schatz  4 ,  Stephen Lee  5
Affiliations
  • 1. Sylvester Comprehensive Cancer Center, Miller School of Medicine, University of Miami, Miami, FL 33136, USA; Division of Hematology, Department of Medicine, Miller School of Medicine, University of Miami, Miami, FL 33136, USA. Electronic address: [email protected].
  • 2. Sylvester Comprehensive Cancer Center, Miller School of Medicine, University of Miami, Miami, FL 33136, USA; Medical Scientist Training Program, Miller School of Medicine, University of Miami, Miami, FL 33136, USA; Molecular and Cellular Pharmacology Graduate Program, Miller School of Medicine, University of Miami, Miami, FL 33136, USA.
  • 3. Bioinformatics Solutions Inc., Waterloo, ON N2L 6J2, Canada.
  • 4. Sylvester Comprehensive Cancer Center, Miller School of Medicine, University of Miami, Miami, FL 33136, USA; Division of Hematology, Department of Medicine, Miller School of Medicine, University of Miami, Miami, FL 33136, USA. Electronic address: [email protected].
  • 5. Sylvester Comprehensive Cancer Center, Miller School of Medicine, University of Miami, Miami, FL 33136, USA; Department of Biochemistry and Molecular Biology, Miller School of Medicine, University of Miami, Miami, FL 33136, USA. Electronic address: [email protected].
PMID: 36595908 DOI: 10.1016/j.xpro.2022.101919
Abstract

Here, we present a protocol using MATRIX (mass spectrometry analysis of active translation factors using ribosome density fractionation and isotopic labeling experiments) platform to investigate changes of the protein synthesis machinery in U87MG glioblastoma cells in response to the rocaglate silvestrol. This protocol describes steps to perform SILAC (stable isotope labeling by Amino acids in Cell Culture), ribosome density fractionation, protein isolation, and mass spectrometry analysis. This approach can be applied to study any adaptive remodeling of protein synthesis machineries. For complete details on the use and execution of this protocol, please refer to Ho et al. (2021).1.

Keywords
Cancer; Mass Spectrometry; Protein Biochemistry; Proteomics.
Products