Ligand-protein target screening from cell matrices using reactive desorption electrospray ionization-mass spectrometry via a native-denatured exchange approach.

Native mass spectrometry has been recognized as a powerful tool for probing interactions between small molecules, such as drugs and natural products, and target proteins. However, the presence of heterogeneous proteins and metabolites in real biological systems can alter the conformations of target proteins or compete with candidate ligands, thus necessitating a method for measuring binding stoichiometries in matrices aside from the extensively used pure/recombinant protein systems. Furthermore, some small molecule-protein interactions have a transient and low-affinity nature and thus can be mis-assigned as nonspecific binding complexes that are often formed during the native ESI process. A native-denatured exchange (NDX) approach was recently developed using a reactive desorption electrospray ionization-mass spectrometer (DESI-MS) setup to screen specific interacting partners. The method works by gradually increasing the composition of denaturing solvents contained in the DESI spray and thus conferring a switch from a native to denatured ionization environment. This change impairs three-dimensional structures of target proteins and disrupts specific ligand-protein interactions, leading to decreased holo/apo ratios. In contrast, ligand-protein complexes exhibiting different trends are assigned as nonspecific interactions. Herein, we applied the NDX approach to probe specific ligand-protein interactions in biological matrices. We first used mixtures of model ligands and proteins to examine the use of reactive DESI-MS in recognizing ligand-target binding in mixtures. Subsequently, we used the NDX approach to analyze binding affinity curves of ligands to target proteins spiked in cell lysates with the aid of size exclusion chromatography and demonstrated its use in probing specific ligand-protein interactions from cell matrices.

Evaluation of Untargeted Metabolomic Strategy for the Discovery of Biomarker of Breast Cancer.

Discovery of disease biomarker based on untargeted metabolomics is informative for pathological mechanism studies and facilitates disease early diagnosis. Numerous of metabolomic strategies emerge due to different sample properties or experimental purposes, thus, methodological evaluation before sample analysis is essential and necessary. In this study, sample preparation, data processing procedure and metabolite identification strategy were assessed aiming at the discovery of biomarker of breast cancer. First, metabolite extraction by different solvents, as well as the necessity of vacuum-dried and re-dissolution, was investigated. The extraction efficiency was assessed based on the number of eligible components (components with MS/MS data acquired), which was more reasonable for metabolite identification. In addition, a simplified data processing procedure was proposed involving the OPLS-DA, primary screening for eligible components, and secondary screening with constraints including VIP, fold change and p value. Such procedure ensured that only differential candidates were subjected to data interpretation, which greatly reduced the data volume for database search and improved analysis efficiency. Furthermore, metabolite identification and annotation confidence were enhanced by comprehensive consideration of mass and MS/MS errors, isotope similarity, fragmentation match, and biological source confirmation. On this basis, the optimized strategy was applied for the analysis of serum samples of breast cancer, according to which the discovery of differential metabolites highly encouraged the independent biomarkers/indicators used for disease diagnosis and chemotherapy evaluation clinically. Therefore, the optimized strategy simplified the process of differential metabolite exploration, which laid a foundation for biomarker discovery and studies of disease mechanism.

Probing specific ligand-protein interactions by native-denatured exchange mass spectrometry.

Probing ligand-target protein interactions provides essential information for deep understanding of biochemical machinery and design of drug screening assays. Native electrospray ionization-mass spectrometry (ESI-MS) is promising for direct analysis of ligand-protein complexes. However, it lacks the ability to distinguish between specific and non-specific ligand-protein interactions, and to further recognize the specifically bound proteins as drug target candidates, which remains as a major challenge in the field of drug developments by far. Herein we report a native-denatured exchange (NDX) mass spectrometry (MS) acquisition approach using a liquid sample-desorption electrospray ionization (LS-DESI) setup, and demonstrate its capability in enabling a change from native detection of noncovalent ligand-protein complexes to denatured analysis using three model ligand-protein complexes including myoglobin, CDP-ribonuclease and N,N',N″-triacetylchitotriose (NAG3)-lysozyme. Notably, we found the NDX-MS approach can readily discriminate specific ligand-protein interactions from nonspecific ones, as revealed by their distinct dynamic profiles of Kd as a function of the DESI spraying flow rate. Consequently, this NDX-MS approach holds promise for future applications to discovering specific protein targets for ligands of interest, and to screening compounds with high specificity to drug targets and thus eliminates off-target effects.