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.

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 A Fast Lysine Cross-linker DOPA Enables Mass Spectrometry Analyses of Protein Unfolding and Weak Protein-protein Interactions

2020 ◽  
Author(s):  
Jian-Hua Wang ◽  
Yu-Liang Tang ◽  
Rohit Jain ◽  
Fan Xiao ◽  
Zhou Gong ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Conformational Changes ◽  
Protein Interactions ◽  
Guanidine Hydrochloride ◽  
Rnase A ◽  
Mass Spectrometry Analysis ◽  
Cross Linking ◽  
Protein Protein Interactions ◽  
Protein Structure Analysis ◽  
Cross Linker

AbstractChemical cross-linking of proteins coupled with mass spectrometry analysis (CXMS) has become a widely used method for protein structure analysis. Central to this technology are chemical cross-linkers. The most popular cross-linkers are N-hydroxysuccinimide (NHS) esters, which react with protein amino groups relatively slowly over 10 minutes or more while in competition with the hydrolysis reaction of NHS esters. To improve the speed of cross-linking, we developed a new class of amine-selective and non-hydrolyzable di-ortho-phthalaldehyde (DOPA) cross-linkers. DOPA can cross-link proteins in 10 seconds under near physiological conditions, which is 60 times faster than the NHS ester cross-linker DSS. DOPA also works at low pH, low temperature, or in the presence of high concentrations of denaturants such as 8 M urea or 6 M guanidine hydrochloride. Further, DOPA-mediated pulse cross-linking captured the dynamic conformational changes associated with RNase A unfolding. Lastly, DOPA outperformed DSS at capturing weak but specific protein-protein interactions.

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