Detecting Substrates of Proteolysis via Silac Assays

• Activity Number: 516
• Type: Contributed
• Date/Time: Wednesday, August 1, 2012 : 10:30 AM to 12:20 PM
• Sponsor: ENAR
• Author(s): William Forrest*+
• Companies: Genentech
• Keywords: proteomics ; mass spectrometry ; robust ; normalization ; apoptosis ; proteolysis

Abstract

SILAC (Stable Isotope Labeling by Amino acids in Cell culture) screens in proteomics study parallel "heavy" and "light" cell cultures. Proteins from the heavy culture contain amino acids engineered to have a higher nominal molecular weight than their counterpart amino acids in the light culture. By tracking paired discrepancies in peptide masses across varying experimental conditions, a range of questions about the fates of proteins within living cells can be explored.

We consider a case study with an NCI-60 colon cancer cell line and an isogenic "daughter" line genetically altered so as to lack the intracellular protein Bax, which is a well-studied regulator of apoptosis. A SILAC assay was conducted to learn about the effects of a known stimulant of apoptosis on the proteins within the cell line both in its wild-type form and in its Bax-deficient daughter line.

We review technologies (e.g, LC-MS/MS) applied to quantify protein peptide levels. Strategies for visualization and normalization are considered. Researchers' questions are addressed using variants of established statistical tools. Links are drawn to prior approaches for other "-omic" data, notably cDNA microarrays.