Thermodynamic Analysis of Protein–Ligand Interactions in Complex Biological Mixtures using a Shotgun Proteomics Approach

Abstract

Shotgun proteomics protocols are widely used for the identification and/or quantitation of proteins in complex biological samples. Described here is a shotgun proteomics protocol that can be used to identify the protein targets of biologically relevant ligands in complex protein mixtures. The protocol combines a quantitative proteomics platform with a covalent modification strategy, termed Stability of Proteins from Rates of Oxidation (SPROX), which utilizes the denaturant dependence of hydrogen peroxide-mediated oxidation of methionine side chains in proteins to assess the thermodynamic properties of proteins and protein-ligand complexes. The quantitative proteomics platform involves the use of isobaric mass tags and a methionine-containing peptide enhancement strategy. The protocol is evaluated in a ligand binding experiment designed to identify the proteins in a yeast cell lysate that bind the well-known enzyme co-factor, β-nicotinamide adenine dinucleotide (NAD+). The protocol is also used to investigate the protein targets of resveratrol, a biologically active ligand with less well-understood protein targets. A known protein target of resveratrol, cytosolic aldehyde dehydrogenase, was identified in addition to six other potential new proteins targets including four that are associated with the protein translation machinery, which has previously been implicated as a target of resveratrol.

Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC)-Based Strategy for Proteome-Wide Thermodynamic Analysis of Protein-Ligand Binding Interactions

“…five times the number of peptide and proteins assayed in Gel Experiment 2, Solution Experiment 1B, or in Solution Experiment 2). This is ϳthree-fivefold more peptide and protein coverage than that previously reported in SPROX experiments using an isobaric mass tagging strategy (16). It is also noteworthy that the solution and gel-based protocols were to some degree complementary in their peptide and protein coverage.…”

“…Two energetics-based approaches, the stability of proteins from rates of oxidation (SPROX) 1 (10,16,17) and pulse proteolysis techniques (13,18), have shown promise for protein-ligand binding analyses on the proteomic scale, but so far have been limited in their proteomic coverage. Although the pulse pro-teolysis technique does utilize targeted mass spectrometrybased proteomics analyses for the identification of hit proteins, the technique relies on gel-based strategies for the resolution, detection, and quantitation of potential protein targets (13,18).…”

“…This reliance on gel-based strategies for protein resolution, detection, and quantitation, ultimately limits the complexity of protein samples that can be interrogated for ligand binding. In contrast, the SPROX technique has been interfaced with conventional bottom-up shotgun proteomics platforms that exploit the capabilities of modern LC-MS/MS systems to resolve, detect, and quantify the protein components of complex biological mixtures (10,16,17).…”

Thermodynamic Analysis of the Geldanamycin–Hsp90 Interaction in a Whole Cell Lysate Using a Mass Spectrometry-Based Proteomics Approach

“…The SPROX technique is a mass spectrometry-based approach for measuring the thermodynamic stability of proteins and protein-ligand complexes [26,27,29,30]. The technique has been previously validated using a series of different model systems, which have included both purified and unpurified proteins and protein-ligand complexes [27,29,30]. Of particular significance to the present study is that the SPROX technique does not require any special labeling of the ligand and that the technique is amenable to the thermodynamic analysis of protein-ligand binding reaction in whole cell lysates.…”

Screening of the Binding of Small Molecules to Proteins by Desorption Electrospray Ionization Mass Spectrometry Combined with Protein Microarray

“…Therefore, the determination of the binding of small molecules to proteins has attracted considerable research attention in recent years [2][3][4]. Owing to large synthetic and natural product compound libraries becoming more easily accessible, the availability of efficient methods for the rapid screening of the binding of these compounds to proteins becomes imperative.…”