Photoaffinity labeling combined with mass spectrometric approaches as a tool for structural proteomics

Abstract

Protein chemistry, such as crosslinking and photoaffinity labeling, in combination with modern mass spectrometric techniques, can provide information regarding protein-protein interactions beyond that normally obtained from protein identification and characterization studies. While protein crosslinking can make tertiary and quaternary protein structure information available, photoaffinity labeling can be used to obtain structural data about ligand-protein interaction sites, such as oligonucleotide-protein, drug-protein and protein-protein interaction. In this article, we describe mass spectrometry-based photoaffinity labeling methodologies currently used and discuss their current limitations. We also discuss their potential as a common approach to structural proteomics for providing 3D information regarding the binding region, which ultimately will be used for molecular modeling and structure-based drug design.

Figure 1

General schema of photolabeling-mass spectrometry experiments. (A) Following interaction of the protein of interest with a photolabeled ligand, the complex is covalently linked by UV irradiation. Isolation of the covalent complex followed by MS allows for identification of binding protein, determination of the ligand:protein stoichiometry, and identification and sequencing of the ligand-binding region of the protein. We used this approach to localize the binding site for an inhibitor of the HIV-1 integrase protein. (B) Alternatively, if the receptor protein is known, the purified protein may be subjected to direct LC-MS following ligand binding, crosslinking and tryptic digestion. This approach may be advantageous when dealing with hydrophobic ligands that may be lost during HPLC separation. HPLC: High-performance liquid chomatography; LC: Liquid chromatography; MALDI: Matrix-assisted laser desorption/ionization; MS: Mass spectrometry; UV: Ultraviolet.

Figure 2

Deconvoluted ESI-MS spectra of (left panel) HIV-integrase and (right panel) HIV-integrase after photoaffinity labeling with the HIV-integrase inhibitor coumarin (R61). (A): the base peak at m/z 19,954 agrees precisely with the MW of HIV-integrase as calculated from the amino acid sequence. The additional peaks at higher m/z can be assigned to oxidation products and β-mercaptoethanol adducts. (B): due to the presence of the photoaffinity label, the peaks are shifted to higher m/z values by 516 Da, which agrees precisely with the expected mass increment for addition of one coumarin molecule. ESI: Electrospray ionization; MS: Mass spectrometry; MW: Molecular weight.

Figure 3

Model of a single HIV integrase dimer rendered by helix. Yellow represents the inhibitor binding region peptide ¹²⁸AACWWAGIK¹³⁶. Blue represents one molecule of the coumarin inhibitor complexed within the binding site determined by PAL-MS. Cyan represents the integrase active site residues (DDE motif) and the sphere is Mg²⁺ chelating at this site. MS: Mass spectrometry; PAL: Photoaffinity labeling.