scholarly journals Structure, bonding, and photoaffinity labeling applications of dialkyldiazirines

Synlett ◽  
2021 ◽  
Author(s):  
Tianyi Zhang ◽  
Alison Ondrus
Keyword(s):  
Mass Spectrometry ◽  
Small Molecule ◽  
Protein Interactions ◽  
Photoaffinity Labeling ◽  
Mass Spectrometry Analysis ◽  
Practical Application ◽  
Basic Principles ◽  
Spectrometry Analysis ◽  
Photoaffinity Probes ◽  
Photoaffinity Crosslinking

Dialkyldiazirine photoaffinity probes are unparalleled tools for the study of small molecule-protein interactions. Here we summarize the basic principles of structure, bonding, and photoreactivity of dialkyldiazirines, current methods for their synthesis, and their practical application in photoaffinity labeling experiments. We demonstrate the unique utility of dialkyldiazirine probes in the context of our recent photoaffinity crosslinking-mass spectrometry analysis to reveal a hidden cholesterol binding site in the Hedgehog morphogen proteins.

Label-free quantitative proteomic analysis of the YAP/TAZ interactome

2014 ◽  
Vol 306 (9) ◽  
pp. C805-C818 ◽  
Author(s):  
Priyanka Kohli ◽  
Malte P. Bartram ◽  
Sandra Habbig ◽  
Caroline Pahmeyer ◽  
Tobias Lamkemeyer ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Protein Interactions ◽  
Affinity Purification ◽  
Single Copy ◽  
Single Step ◽  
Mass Spectrometry Analysis ◽  
Single Shot ◽  
Label Free ◽  
Protein Protein Interactions ◽  
Spectrometry Analysis

The function of an individual protein is typically defined by protein-protein interactions orchestrating the formation of large complexes critical for a wide variety of biological processes. Over the last decade the analysis of purified protein complexes by mass spectrometry became a key technique to identify protein-protein interactions. We present a fast and straightforward approach for analyses of interacting proteins combining a Flp-in single-copy cellular integration system and single-step affinity purification with single-shot mass spectrometry analysis. We applied this protocol to the analysis of the YAP and TAZ interactome. YAP and TAZ are the downstream effectors of the mammalian Hippo tumor suppressor pathway. Our study provides comprehensive interactomes for both YAP and TAZ and does not only confirm the majority of previously described interactors but, strikingly, revealed uncharacterized interaction partners that affect YAP/TAZ TEAD-dependent transcription. Among these newly identified candidates are Rassf8, thymopoetin, and the transcription factors CCAAT/enhancer-binding protein (C/EBP)β/δ and core-binding factor subunit β (Cbfb). In addition, our data allowed insights into complex stoichiometry and uncovered discrepancies between the YAP and TAZ interactomes. Taken together, the stringent approach presented here could help to significantly sharpen the understanding of protein-protein networks.

scholarly journals Optimization of Formaldehyde Cross-Linking for Protein Interaction Analysis of Non-Tagged Integrinβ1

2010 ◽  
Vol 2010 ◽  
pp. 1-13 ◽  
Author(s):  
Cordula Klockenbusch ◽  
Juergen Kast
Keyword(s):  
Mass Spectrometry ◽  
Protein Interactions ◽  
Interaction Analysis ◽  
Protein Complexes ◽  
Human Platelets ◽  
Jurkat Cells ◽  
Mass Spectrometry Analysis ◽  
Cross Linking ◽  
High Molecular Weight Complex ◽  
Spectrometry Analysis

Formaldehyde cross-linking of protein complexes combined with immunoprecipitation and mass spectrometry analysis is a promising technique for analysing protein-protein interactions, including those of transient nature. Here we used integrinβ1 as a model to describe the application of formaldehyde cross-linking in detail, particularly focusing on the optimal parameters for cross-linking, the detection of formaldehyde cross-linked complexes, the utility of antibodies, and the identification of binding partners. Integrinβ1 was found in a high molecular weight complex after formaldehyde cross-linking. Eight different anti-integrinβ1 antibodies were used for pull-down experiments and no loss in precipitation efficiency after cross-linking was observed. However, two of the antibodies could not precipitate the complex, probably due to hidden epitopes. Formaldehyde cross-linked complexes, precipitated from Jurkat cells or human platelets and analyzed by mass spectrometry, were found to be composed of integrinβ1,α4 andα6 orβ1,α6,α2, andα5, respectively.

Prediction and collection of protein–metabolite interactions

2021 ◽  
Author(s):  
Tianyi Zhao ◽  
Jinxin Liu ◽  
Xi Zeng ◽  
Wei Wang ◽  
Sheng Li ◽  
...  
Keyword(s):  
Small Molecule ◽  
Protein Interactions ◽  
Large Scale ◽  
Rapid Development ◽  
Area Under The Curve ◽  
Membrane Receptors ◽  
Mass Spectrometry Analysis ◽  
Spectrometry Analysis ◽  
Cellular Processes ◽  
The Rich

Abstract Interactions between proteins and small molecule metabolites play vital roles in regulating protein functions and controlling various cellular processes. The activities of metabolic enzymes, transcription factors, transporters and membrane receptors can all be mediated through protein–metabolite interactions (PMIs). Compared with the rich knowledge of protein–protein interactions, little is known about PMIs. To the best of our knowledge, no existing database has been developed for collecting PMIs. The recent rapid development of large-scale mass spectrometry analysis of biomolecules has led to the discovery of large amounts of PMIs. Therefore, we developed the PMI-DB to provide a comprehensive and accurate resource of PMIs. A total of 49 785 entries were manually collected in the PMI-DB, corresponding to 23 small molecule metabolites, 9631 proteins and 4 species. Unlike other databases that only provide positive samples, the PMI-DB provides non-interaction between proteins and metabolites, which not only reduces the experimental cost for biological experimenters but also facilitates the construction of more accurate algorithms for researchers using machine learning. To show the convenience of the PMI-DB, we developed a deep learning-based method to predict PMIs in the PMI-DB and compared it with several methods. The experimental results show that the area under the curve and area under the precision-recall curve of our method are 0.88 and 0.95, respectively. Overall, the PMI-DB provides a user-friendly interface for browsing the biological functions of metabolites/proteins of interest, and experimental techniques for identifying PMIs in different species, which provides important support for furthering the understanding of cellular processes. The PMI-DB is freely accessible at http://easybioai.com/PMIDB.

scholarly journals Optimizing the fragment complementation of APEX2 for detection of specific protein-protein interactions in live cells

2017 ◽  
Vol 7 (1) ◽  
Author(s):  
Miaomiao Xue ◽  
Junjie Hou ◽  
Linlin Wang ◽  
Dongwan Cheng ◽  
Jingze Lu ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Protein Interactions ◽  
High Specificity ◽  
Mass Spectrometry Analysis ◽  
Live Cells ◽  
Protein Protein Interactions ◽  
Hek 293 ◽  
Spectrometry Analysis ◽  
Specificity And Sensitivity ◽  
Fragment Complementation

Abstract Dynamic protein-protein interactions (PPIs) play crucial roles in cell physiological processes. The protein-fragment complementation (PFC) assay has been developed as a powerful approach for the detection of PPIs, but its potential for identifying protein interacting regions is not optimized. Recently, an ascorbate peroxidase (APEX2)-based proximity-tagging method combined with mass spectrometry was developed to identify potential protein interactions in live cells. In this study, we tested whether APEX2 could be employed for PFC. By screening split APEX2 pairs attached to FK506-binding protein 12 (FKBP) and the FKBP12-rapamycin binding (FRB) domain, which interact with each other only in the presence of rapamycin, we successfully obtained an optimized pair for visualizing the interaction between FRB and FKBP12 with high specificity and sensitivity in live cells. The robustness of this APEX2 pair was confirmed by its application toward detecting the STIM1 and Orial1 homodimers in HEK-293 cells. With a subsequent mass spectrometry analysis, we obtained five different biotinylated sites that were localized to the known interaction region on STIM1 and were only detected when the homodimer formed. These results suggest that our PFC pair of APEX2 provides a potential tool for detecting PPIs and identifying binding regions with high specificity in live cells.

scholarly journals A New Drug Discovery Approach Based on Thermal Proteome Profiling to Develop More Effective Drugs

2021 ◽  
Vol 8 (2) ◽  
Author(s):  
Elham Gholizadeh ◽  
Mostafa Rezaei-Tavirani ◽  
Alireza Emadi ◽  
Reza Karbalaei ◽  
Ali Khaleghian
Keyword(s):  
Mass Spectrometry ◽  
Protein Interactions ◽  
Large Scale ◽  
Protein Extraction ◽  
Mass Spectrometry Analysis ◽  
Structural Stabilization ◽  
Proteome Profiling ◽  
Spectrometry Analysis ◽  
Drug Concentrations ◽  
Low Efficiency

: The search for disease-related targets and studying drug-protein and protein-protein interactions are central issues that would accelerate the clinical approval of a drug. Also, by developing an accurate method in this regard, time and resource consumption will significantly decrease. The low efficiency of some drugs in humans is a grave issue leading to a low rate of FDA approval after spending billions of dollars and decades of research. Several strategies and methods have been expanded to fill this gap, such as drug affinity responsive target stability (DARTS), stability of proteins from rates of oxidation (SPROX), cellular thermal shift assay (CETSA), and finally, thermal proteome profiling (TPP). The TPP is based on the combination of CETSA and quantitative mass spectrometry. Among recently introduced proteomics technologies, TPP demonstrates the ability to offer detailed proteomic profiles for the large-scale analysis of protein-ligand interactions, including endogenous ligands and proteins like cofactors and metabolites. TPP facilitates the identification of the markers governing drug efficacy and toxicity and provides an unbiased measure for estimating the rate of drug-target engagement. At a glance at TPP steps, after protein extraction, the molecule is exposed to different temperatures and drug concentrations. After discarding solubilized and stabilized proteins, the protein’s identity is investigated by mass spectrometry analysis. As a result of the protein’s structural stabilization after binding to its substrate, TTP helps to accurately identify target proteins with high throughput. In this study, we aimed to introduce the basics of this method and review most recent studies on this technique.

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