Native Mass Spectrometry Analysis of Affinity-Captured Endogenous Yeast RNA Exosome Complexes

2019 ◽  
pp. 357-382 ◽  
Author(s):  
Paul Dominic B. Olinares ◽  
Brian T. Chait
Keyword(s):  
Mass Spectrometry ◽  
Native Mass Spectrometry ◽  
Mass Spectrometry Analysis ◽  
Spectrometry Analysis ◽  
Rna Exosome

scholarly journals Molecular insights into mechanisms of GPCR hijacking by Staphylococcus aureus

2021 ◽  
Vol 118 (42) ◽  
pp. e2108856118
Author(s):  
Claire M. Grison ◽  
Paul Lambey ◽  
Sylvain Jeannot ◽  
Elise Del Nero ◽  
Simon Fontanel ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Staphylococcus Aureus ◽  
G Protein ◽  
Protein Complexes ◽  
Native Mass Spectrometry ◽  
Mass Spectrometry Analysis ◽  
Spectrometry Analysis ◽  
Exchange Mass Spectrometry ◽  
Toxin Binding ◽  
And Function

Atypical chemokine receptor 1 (ACKR1) is a G protein–coupled receptor (GPCR) targeted by Staphylococcus aureus bicomponent pore-forming leukotoxins to promote bacterial growth and immune evasion. Here, we have developed an integrative molecular pharmacology and structural biology approach in order to characterize the effect of leukotoxins HlgA and HlgB on ACKR1 structure and function. Interestingly, using cell-based assays and native mass spectrometry, we found that both components HlgA and HlgB compete with endogenous chemokines through a direct binding with the extracellular domain of ACKR1. Unexpectedly, hydrogen/deuterium exchange mass spectrometry analysis revealed that toxin binding allosterically modulates the intracellular G protein–binding domain of the receptor, resulting in dissociation and/or changes in the architecture of ACKR1−Gαi1 protein complexes observed in living cells. Altogether, our study brings important molecular insights into the initial steps of leukotoxins targeting a host GPCR.

Performing native mass spectrometry analysis on therapeutic antibodies

Methods ◽  
2014 ◽  
Vol 65 (1) ◽  
pp. 11-17 ◽  
Author(s):  
Natalie J. Thompson ◽  
Sara Rosati ◽  
Albert J.R. Heck
Keyword(s):  
Mass Spectrometry ◽  
Native Mass Spectrometry ◽  
Mass Spectrometry Analysis ◽  
Therapeutic Antibodies ◽  
Spectrometry Analysis

scholarly journals Combinatorial native MS and LC-MS/MS approach reveals high intrinsic phosphorylation of human Tau but minimal levels of other key modifications

2020 ◽  
Author(s):  
Friedel Drepper ◽  
Jacek Biernat ◽  
Senthillvelrajan Kaniyappan ◽  
Helmut E. Meyer ◽  
Eva Maria Mandelkow ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Posttranslational Modifications ◽  
Native Mass Spectrometry ◽  
Eukaryotic Cell ◽  
Cell System ◽  
Mass Spectrometry Analysis ◽  
Phosphorylation Sites ◽  
Spectrometry Analysis ◽  
Phosphorylated Peptides ◽  
Tau Aggregation

AbstractAbnormal changes in the neuronal microtubule-associated protein Tau, such as hyperphosphorylation and aggregation, are considered hallmarks of cognitive deficits in Alzheimer disease. Hyperphosphorylation is thought to take place before aggregation, and therefore it is often assumed that phosphorylation predisposes Tau towards aggregation. However, the nature and extent of phosphorylation has remained ill-defined. Tau protein contains up to 85 potential phosphorylation sites (80 Ser/Thr, and 5 Tyr P-sites), many of which can be phosphorylated by various kinases because the unfolded structure of Tau makes them accessible. However, limitations in methods (e.g. in mass spectrometry of phosphorylated peptides, or antibodies against phospho-epitopes) have led to conflicting results regarding the overall degree of phosphorylation of Tau in cells. Here we present results from a new approach, that is based on native mass spectrometry analysis of intact Tau expressed in a eukaryotic cell system (Sf9) which reveals Tau in different phosphorylation states. The extent of phosphorylation is remarkably heterogeneous with up to ∼20 phosphates (Pi) per molecule and distributed over 51 sites (including all P-sites published so far and additional 18 P-sites). The medium phosphorylated fraction Pm showed overall occupancies centered at 8 Pi (± 5 Pi) with a bell-shaped distribution, the highly phosphorylated fraction Ph had 14 Pi (± 6 Pi). The distribution of sites was remarkably asymmetric (with 71% of all P-sites located in the C-terminal half of Tau). All phosphorylation sites were on Ser or Thr residues, but none on Tyr. Other known posttranslational modifications of Tau were near or below our detection limit (e.g. acetylation, ubiquitination). None of the Tau fractions self-assemble readily, arguing that Tau aggregation is not promoted by phosphorylation per se but requires additional factors.

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