Dataset Information

SILAC-Based Strategy for Proteome-Wide Analysis of Protein-Ligand Binding Interactions (Part 1/2)

ABSTRACT: A quantitative mass spectrometry-based proteomics method for the large-scale thermodynamic analysis of protein-ligand binding interactions is described here. The methodology utilizes a chemical modification strategy termed, Stability of Proteins from Rates of Oxidation (SPROX), in combination with a Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) approach to compare the equilibrium folding/unfolding properties of proteins in the absence and presence of target ligands. The method, which is general with respect to ligand, measures the ligand-induced changes in protein stability associated with protein-ligand binding. The methodology is demonstrated in a proof-of-principle study in which the well-characterized protein-drug interaction between cyclosporine A and cyclophilin A is analyzed in the context of a yeast cell lysate. The method is also utilized for the global analysis of adenosine triphosphate (ATP) binding to proteins in the yeast proteome using the non-hydrolyzable ATP analogue, adenylyl imidodiphosphate (AMP-PNP), and the proteins in a yeast cell lysate.

INSTRUMENT(S): 6520 Quadrupole Time-of-Flight LC/MS

ORGANISM(S): Saccharomyces Cerevisiae (strain Atcc 204508 / S288c) (baker's Yeast)

SUBMITTER: Jagat Adhikari

LAB HEAD: Michael C. Fitzgerald

PROVIDER: PXD000858 | Pride | 2014-07-31

REPOSITORIES: Pride

ACCESS DATA

Dataset's files

Source:

			Action	DRS
	SILACSPROXCnBRATP25to30kDa.ssv	Other
	SILACSPROXCnBr12to15K.ssv	Other
	SILACSPROXCnBr15to18K.ssv	Other
	SILACSPROXCnBr37to50.ssv	Other
	SILACSPROXCnBr50to75.ssv	Other

Items per page:

1 - 5 of 268

Publications

StableIsotope Labeling with Amino Acids in Cell Culture (SILAC)-based strategy for proteome-wide thermodynamic analysis of protein-ligand binding interactions.

Tran Duc T DT Adhikari Jagat J Fitzgerald Michael C MC

Molecular & cellular proteomics : MCP 20140416 7

Described here is a quantitative mass spectrometry-based proteomics method for the large-scale thermodynamic analysis of protein-ligand binding interactions. The methodology utilizes a chemical modification strategy termed, Stability of Proteins from Rates of Oxidation (SPROX), in combination with a Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) approach to compare the equilibrium folding/unfolding properties of proteins in the absence and presence of target ligands. The method ...[more]

PMID: 24741112

Similar Datasets

Project description:Large-scale studies of protein phosphorylation have generally focused on determining the sites and stoichiometry of phosphorylated proteins derived from different biological specimens such as cell lines and tissue samples. While such information is important for understanding the role of phosphorylation in biological systems, it fails to expose the relationship between protein phosphorylation and protein function. Because of the close link between protein function and protein folding stability, knowledge about phosphorylation-induced protein folding stability changes can lead to a better understanding of the functional effects of protein phosphorylation. As part of this work, the Stability of Proteins from Rate of OXidation (SPROX) and Limited Proteolysis (LiP) techniques were utilized to identify phosphorylation-induced conformational and stability changes in proteins derived from a human breast cancer cell line MCF-7. This was accomplished by comparing the conformation properties of proteins in two MCF-7 cell lysates including one that was and one that was not dephosphorylated with alkaline phosphatase. The SPROX technique, which probes the global unfolding/refolding properties of proteins, identified 168 proteins with dephosphorylation-induced stability changes, and the LiPs technique, which probes the more local unfolding/refolding properties of proteins, identified 251 proteins with dephosphorylation-induced stability changes. A total of 49 proteins identified here with dephosphorylation-induced conformational changes, were previously identified in phosphoproteomic studies to be differentially phosphorlyated in different human breast cancer subtypes. A total of 98 proteins identified here overlapped with protein hits previously found to be differentially stabilized in four human breast cancer phenotypes, suggesting that the phosphorylated changes observed here may be disease related. The experimental approach and results reported here create a novel approach for identifying functionally significant PTMs in proteins.