By using a limited-proteolysis strategy that employs a large amount of trypsin to generate peptides directly from native proteins, we found that ligand-induced protein local stability shifts can be sensitively detected on a proteome-wide scale. This enabled us to develop the peptide-centric local stability assay, a modification-free approach that achieves unprecedented sensitivity in proteome-wide target identification and binding-region determination. We demonstrate the broad applications of the peptide-centric local stability assay by investigating interactions across various biological contexts.
View Article and Find Full Text PDFUnlabelled: The potential of CRISPR/Cas systems for nucleic acid detection in novel biosensing applications is remarkable. The current clinical diagnostic detection of () is based on serological identification, culture, and PCR. We report a rapid, simple, and sensitive method for detecting and screening for .
View Article and Find Full Text PDFAdverse health outcomes caused by environmental chemicals are often initiated via their interactions with proteins. Essentially, one environmental chemical may interact with a number of proteins and/or a protein may interact with a multitude of environmental chemicals, forming an intricate interaction network. Omics-wide protein-environmental chemical interaction profiling (PECI) is of prominent importance for comprehensive understanding of these interaction networks, including the toxicity mechanisms of action (MoA), and for providing systematic chemical safety assessment.
View Article and Find Full Text PDFTarget proteins are often stabilized after binding with a ligand and thereby typically become more resistant to denaturation. Based on this phenomenon, several methods without the need to covalently modify the ligand have been developed to identify target proteins for a specific ligand. These methods usually employ complicated workflows with high cost and limited throughput.
View Article and Find Full Text PDFExisting thermal shift-based mass spectrometry approaches are able to identify target proteins without chemical modification of the ligand, but they are suffering from complicated workflows with limited throughput. Herein, we present a new thermal shift-based method, termed matrix thermal shift assay (mTSA), for fast deconvolution of ligand-binding targets and binding affinities at the proteome level. In mTSA, a sample matrix, treated horizontally with five different compound concentrations and vertically with five technical replicates of each condition, was denatured at a single temperature to induce protein precipitation, and then, data-independent acquisition was employed for quick protein quantification.
View Article and Find Full Text PDFThe process of protein precipitation can be used to decipher the interaction of ligand and protein. For example, the classic Thermal Proteome Profiling (TPP) method uses heating as the driving force for protein precipitation, to discover the drug target protein. Under heating or other denature forces, the target protein that binds with the drug compound will be more resistant to precipitation than the free protein.
View Article and Find Full Text PDFCell surface is the primary site for sensing extracellular stimuli. The knowledge of the transient changes on the surfaceome upon a perturbation is very important as the initial changed proteins could be driving molecules for some phenotype. In this study, we report a fast cell surface labeling strategy based on peroxidase-mediated oxidative tyrosine coupling strategy, enabling efficient and selective cell surface labeling within seconds.
View Article and Find Full Text PDFMAX and MXene have received considerable attention owing to their outstanding performance in fields like battery and catalysis. However, their possible biomedical applications have rarely been considered, especially the affinity chromatographic applications in proteomics. In this work, considering the large number of exposed metal sites, small binding potential resistance and fast mass transfer speed, layered ternary carbides MAX-TiAlC and MXene-TiC with a two-dimensional nanostructure were successfully explored for the first time as affinity chromatography stationary phases for the specific capture of phosphopeptides from complex biological samples.
View Article and Find Full Text PDFWhile thermal proteome profiling (TPP) shines in the field of drug target screening by analyzing the soluble fraction of the proteome samples treated at high temperature, the counterpart, the insoluble precipitate, has been overlooked for a long time. The analysis of the precipitate is hampered by the inefficient sample processing procedure. Herein, we propose a novel method, termed microparticle-assisted precipitation screening (MAPS), for drug target identification.
View Article and Find Full Text PDFAlthough capillary liquid chromatography married with tandem mass spectrometry (cLC-MS/MS) has become a powerful technique for proteomics and metabolomics research, it is still a great challenge to fabricate durable capillary-based analytical columns coupling continuous nanoflow (<1 000 nL/min) electrospray ionization (ESI) with MS, owing to the issue of clogging and fragile of emitters. Here, we proposed a simple approach to integrate microstructured photonic fibers (MPFs) into wide bore capillaries with 150 μm i.d.
View Article and Find Full Text PDFIn this study, we presented an enrichment-free approach for the sensitive analysis of protein phosphorylation in minute amounts of samples, such as purified protein complexes. This method takes advantage of the high sensitivity of parallel reaction monitoring (PRM). Specifically, low confident phosphopeptides identified from the data-dependent acquisition (DDA) data set were used to build a pseudotargeted list for PRM analysis to allow the identification of additional phosphopeptides with high confidence.
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