OCT 12, 2017 9:00 AM PDT

Profiling the cell surface of cancer cell lines for biomarker/drug target discovery using MS-based proteomics

Speaker
  • Senior Scientist, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research (FNLCR)
    BIOGRAPHY

Abstract

In order to expand the treatment options of cancers driven by oncogenic RAS, new cell surface targets need to be identified and characterized. Here, we describe mass spectrometry based phenotyping of the KRASG12V cell surface using MCF10A-KRASG12V as a cell line model of constitutively activated KRAS. Extensive view of the MCF10A-KRASG12V surface proteome was achieved by applying concurrently targeted hydrazide-based cell surface capturing (CSC) technology and global shotgun membrane (GSM) proteomics. Our combined approach revealed 666 plasma membrane proteins unique to the MCF10A-KRASG12V cell surface. Of these, 104 were cell surface glycoproteins identified by CSC technology while 562 cell surface proteins were identified using GSM proteomics. K-Ras was exclusively identified by GSM proteomics in the membrane fraction of MCF10A-KRASG12V cells and subsequently cross-validated by Western blot (WB) analysis. Subtractive proteomics, spectral counting-based based quantitation of changes in protein abundances, and Ingenuity Pathway Analysis showed that this investigation reliably revealed expected K-Ras induced changes at the surface of MCF10A-KRASG12V cells as well as alterations in cell surface protein expression with very little prior information. This analysis uncovered a subset of eight proteins identified exclusively at the cell surface of MCF10A-KRASG12V cells by both CSC and GSM technologies. Of these, two were further cross-validated using immunofluorescence and WB analysis. Furthermore, scanning electron microscopy and functional cell assays showed extensive changes at the KRASG12V cell surface consistent with widespread epithelial to mesenchymal transformation (EMT). Taken together, this dataset greatly extends the known molecular phenotype of the MCF10A-KRASG12V cell surface and reveals the important role of EMT in the pathophysiology of the KRASG12V driven malignant transformation in MCF10A- KRASG12V model cell line.


Show Resources
You May Also Like
MAY 17, 2022 9:00 AM PDT
MAY 17, 2022 9:00 AM PDT
Date: May 17, 2022 Time: 9:00am (PDT), 12:00pm (EDT), 8:00pm (CEST) Gene therapeutics have great potential to treat many severe diseases in an unprecedented, targeted manner. The biopharmace...
JUN 28, 2022 7:00 AM PDT
JUN 28, 2022 7:00 AM PDT
Date: June 28, 2022 Time: 3:00pm (BST), 4:00pm (CET), 9:00am (CST), 7am (PST) Light-sheet microscopy is an extremely versatile imaging technique with a vast range of implementations that are...
JUN 21, 2022 6:00 AM PDT
JUN 21, 2022 6:00 AM PDT
Date: June 21, 2022 Time: 6:00am (PDT), 9:00am (EDT), 3:00pm (CEST) The global understanding and practice of medicine is currently undergoing a revolutionary change. This shift to precision...
AUG 24, 2022 7:00 AM PDT
AUG 24, 2022 7:00 AM PDT
Date: August 24, 2022 Time: 7:00am (PDT), 10:00pm (EDT), 4:00pm (CEST) Light field microscopy was first introduced in 2006, and allows users to capture the 4D light field within the microsco...
APR 26, 2022 7:00 AM PDT
C.E. CREDITS
APR 26, 2022 7:00 AM PDT
Date: April 19, 2022 Time: 7:00am (PDT), 10:00am (EDT), 4:00pm (CEST) High-content (HC) phenotypic profiling approaches are a powerful tool to study the effect of biological, genetic, and ch...
SEP 13, 2022 8:00 AM PDT
SEP 13, 2022 8:00 AM PDT
Date: September 13, 2022 Time: 8:00am (PDT), 11:00am (EDT), 5:00pm (CEST) Preparedness against pandemic diseases demands rapid-response vaccine technology and ready-to-use analytical methods...
OCT 12, 2017 9:00 AM PDT

Profiling the cell surface of cancer cell lines for biomarker/drug target discovery using MS-based proteomics



Show Resources
Loading Comments...
Show Resources
Attendees