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

Duc T Tran,Jagat Adhikari,Michael C Fitzgerald

doi:10.1074/mcp.m113.034702

Abstract

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, 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 (CsA) and cyclophilin A was successfully analyzed in the context of a yeast cell lysate. A control experiment was also performed to assess the method's false positive rate of ligand discovery, which was found to be on the order of 0.4 - 3.5%. The new method was utilized to characterize the adenosine triphosphate (ATP)-interactome in Saccharomyces cerevisiae using the nonhydrolyzable ATP analog, adenylyl imidodiphosphate (AMP-PNP), and the proteins in a yeast cell lysate. The new methodology enabled the interrogation of 526 yeast proteins for interactions with ATP using 2035 peptide probes. Ultimately, 325 peptide hits from 139 different proteins were identified. Approximately 70% of the hit proteins identified in this work were not previously annotated as ATP binding proteins. However, nearly two-thirds of the newly discovered ATP interacting proteins have known interactions with other nucleotides and co-factors (e.g. NAD and GTP), DNA, and RNA based on GO-term analyses. The current work is the first proteome-wide profile of the yeast ATP-interactome, and it is the largest proteome-wide profile of any ATP-interactome generated, to date, using an energetics-based method. The data is available via ProteomeXchange with identifiers PXD000858, DOI 10.6019/PXD000858, and PXD000860.

Highlights

From the ‡Department of Biochemistry, Duke University, Medical Center, Durham, North Carolina 27710; §Department of Chemistry, Duke University, Durham, North Carolina 27708
Methionine residues in proteins that were oxidized in the stability of proteins from rates of oxidation (SPROX) analysis are protected from cyanogen bromide (CnBr) cleavage
As proteins are unfolded in the presence of increasing concentrations of the chemical denaturant used in the SPROX analysis, the “buried” methionine residues in a protein’s three-dimensional structure are exposed to solvent, get oxidized in the SPROX analysis, and become protected from cyanogen bromide cleavage

Summary

EXPERIMENTAL PROCEDURES

Yeast Cell Lysate Preparation—A glycerol stock of the Saccharomyces cerevisiae deletion strain BY4739 (Open Biosystems, Lafayette, CO), an auxotroph for lysine, was streaked on a Petri-dish containing synthetic complete (SC) media and 30 mg/L of light L-Lysine. In each SILAC-SPROX experiment the (Ϫ) and (ϩ) ligand stock solutions were equilibrated for 30 – 60 min, before 20 ␮l aliquots of the samples (containing ϳ200 – 400 ␮g of total protein each) were diluted into a series of denaturant containing buffer stock solutions. In each SILAC-SPROX analysis, the (Ϫ) and (ϩ) ligand samples (i.e. the heavy and light yeast cell lysate sample) in buffers containing the same denaturant concentration were combined (see Step 4 in Fig. 1), and the light and heavy-labeled proteins in each sample were precipitated with TCA. Cyanogen Bromide Treatment—For the gel-based experiments performed in the ATP-binding study, the combined (Ϫ) and (ϩ) ligand protein pellets from the SPROX analyses were re-dissolved in 100 ␮l of a 70% (v/v) solution of formic acid (TCI America, Portland, OR). Based on this global analysis and our requirement that hit peptides must have significantly altered L/H ratios at two or more consecutive denaturant concentrations, the estimated p value associated with each peptide hit in this study was Ͻ0.01, except in the case of peptide hits identified in Gel Experiment 2, where the estimated p value associated with each peptide hit was Ͻ0.03

RESULTS

Total peptides

DISCUSSION