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.Expert Rev. Proteomics 3(4), 399-408 (2006) In photoaffinity labeling (PAL), a ligand possessing a UV-photoactivating group is incubated with its receptor to form, first, a noncovalently bound ligand-receptor complex in which the ligand binds specifically to the binding site or pocket in the receptor due to its affinity. Some ligands, such as oligonucleotides, contain intrinsic photoactivating groups; alternatively, these groups can be introduced by synthesis into ligands for peptides and small molecules, such as drugs. It is important to establish that the incorporated photoactivating groups do not significantly alter the binding affinity of the ligand to its receptor and its functionality, compared with the nonderivatized ligand. Subsequent to the complex formation, UV irradiation triggers the photoactivating group in the ligand and leads to the conversion of the noncovalent binding into a covalent link between the ligand and its receptor at the binding site. A covalent ligand-receptor complex makes the study of the complex easier, since it permits the use of more rigorous but harsher analysis tools, such as SDS-PAGE, HPLC and mass spectrometry (MS), which would typically lead to the dissociation of the complex with information regarding the binding sites no longer being obtainable.Photoaffinity labeling with radioactively labeled ligands is a widely used method to determine the competitive binding affinities of other molecules for the binding sites in the binding pocket occupied by the radioactively labeled ligand and, as such, is a valuable tool for drug screening. When photoaffinity techniques are combined with modern MS, this approach can provide additional structural and mechanistic information regarding the protein complex, such as the ligand-receptor stoichiometry, a map of the binding pocket, and even the exact identification of the amino acid residues, which are involved in the ligand-receptor interaction. The data obtained can be used to create a 3D model of the ligand-binding pocket that might provide better insight into the nature of the ligand-receptor interaction. This knowledge can be v...

Photoaffinity Labeling of Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) Targets in Mammalian Cells*

“…2B (lower panel). To narrow down the list of candidates for further study, bands were prioritized that showed Ͼ2-fold reduction in 32 P signal intensity when the PAL reaction was performed in the presence of NAADP (10 M). From this analysis, it was evident that four bands, at ϳ250 kDa (band 1), ϳ102 kDa (band 2), ϳ27 kDa (band 8), and a doublet at 22/23 kDa (band 9), met this criterion.…”

“…For resolution of the 32 P signal, air-dried gels were exposed on phosphor screens (Packard Instrument Co.) at room temperature or on extra-sensitive x-ray film (Thermo Scientific) for 24 -96 h at Ϫ80°C. Quantification of 32 P signals was performed densitometrically using OptiQuant software (Version 3.0, Packard Instrument Co.). Curve fitting and statistical analyses were performed using GraphPad Prism (GraphPad Software Inc.).…”

Probing the Conformation of the ISWI ATPase Domain With Genetically Encoded Photoreactive Crosslinkers and Mass Spectrometry

“…With advances in MS instrumentation and computational analysis of the MS data, it became possible to precisely determine the residues involved in the crosslink (3)(4)(5). Crosslinks provide constraints on the through-space distance of the attachment sites, and this information can aid structure prediction (6,7), can distinguish protein conformations (8), and can identify the interaction surface between proteins (9,10) and between proteins and their ligands (11). Crosslinking-MS-based methods are widely applicable as they only require microgram quantities of protein, are not limited by protein size or solubility and are relatively tolerant against sample heterogeneity.…”

Tools used to study how protein complexes are assembled in signaling cascades

“…Crosslinking reagents are used extensively and in combination with MS, are a very powerful tool. 1,[94][95][96] High throughput screens provide an excellent start point for determining what cohort of proteins interact with the test protein and in combination with bioinformatics and computational tools they will allow the researcher to predict with a high degree of certainty what signaling pathways the test protein is involved in. It is essential then to move to a series of validation systems to confirm an interaction and also to identify the binding interface between the two proteins.…”