In plantachemical cross-linking and mass spectrometry analysis of protein structure and interaction in Arabidopsis

PROTEOMICS ◽  
2016 ◽  
Vol 16 (13) ◽  
pp. 1915-1927 ◽  
Author(s):  
Xinliang Zhu ◽  
Fengchao Yu ◽  
Zhu Yang ◽  
Shichang Liu ◽  
Chen Dai ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Protein Structure ◽  
Mass Spectrometry Analysis ◽  
Cross Linking ◽  
Spectrometry Analysis

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.

scholarly journals Treatment of Microglia with Anti-PrP Antibodies Induces Neuronal Allergenicity

2020 ◽  
Author(s):  
Utpal Kumar Adhikari ◽  
Elif Sakiz ◽  
Umma Habiba ◽  
Sachin Kumar ◽  
Meena Mikhael ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Molecular Mechanisms ◽  
Neuroblastoma Cell ◽  
Mass Spectrometry Analysis ◽  
Cross Linking ◽  
Antibody Treatment ◽  
Spectrometry Analysis ◽  
N2a Cells ◽  
Related Proteins

Abstract Background: Previous reports identified proteins associated with ‘apoptosis’ following cross-linking PrPC with motif-specific anti-PrP antibodies in vivo and in vitro. The molecular mechanisms underlying this IgG-mediated neurotoxicity and the role of the activated proteins in the apoptotic pathways leading to neuronal death has not been properly defined. Previous reports implicated a number of proteins, including apolipoprotein E, cytoplasmic phospholipase A2, prostaglandin and calpain with anti-PrP antibody-mediated ‘apoptosis’, however, these proteins are also known to play an important role in allergy. In this study, we investigated whether cross-linking PrPC with anti-PrP antibodies stimulates a neuronal allergenic response.Methods: Initially, we predicted the allergenicity of the epitope sequences associated with ‘neurotoxic’ anti-PrP antibodies using allergenicity prediction servers. We then investigated whether anti-PrP antibody treatment of neuronal (N2a) and microglia (N11) cell lines leads to a neuronal allergenic response.Results: We found that both tail- and globular-epitopes were allergenic. Specifically, binding regions that contain epitopes for ‘neurotoxic’ antibodies such as ICSM18 (146-159), ICSM35 (91-110), POM 1 (138-147), POM 2 (57-88) and POM 3 (95-100) lead to activation of allergenic related proteins. Following direct application of anti-PrPC antibodies on N2a cells, mass spectrometry analysis identified 4 neuronal allergenic-related proteins when compared with untreated cells. Furthermore, mass spectrometry analysis identified 8 neuronal allergenic-related proteins following cross-linking N11 cells with anti-PrPC antibodies prior to co-culture with N2a cells, when compared with untreated cells. Of importance, we showed that the allergenic effects triggered by the anti-PrP antibodies were more potent when antibody-treated microglia were co-cultured with the neuroblastoma cell line. Furthermore, in both direct and co-culture with antibody-treated microglia, we demonstrate that the allergenic proteome was part of the PrPC-interactome. Conclusions: This study showed for the first time that anti-PrP antibody binding to PrPC triggers a neuronal allergenic response (we termed ‘IgG-Mediated Neuronal Allergenic Toxicity’) and highlights the important role of microglia in triggering IgG-mediated neuronal allergenic toxicity. Moreover, this study provides an important impetus for including allergenic assessment of therapeutic antibodies for neurodegenerative to derive safe and targeted biotherapeutics.

scholarly journals Characterization of the Raptor/4E-BP1 Interaction by Chemical Cross-linking Coupled with Mass Spectrometry Analysis

2014 ◽  
Vol 289 (8) ◽  
pp. 4723-4734 ◽  
Author(s):  
Kimberly Coffman ◽  
Bing Yang ◽  
Jie Lu ◽  
Ashley L. Tetlow ◽  
Emelia Pelliccio ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Mass Spectrometry Analysis ◽  
Cross Linking ◽  
Spectrometry Analysis ◽  
Chemical Cross Linking
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