scholarly journals Mass spectrometry analysis of the photosystem II assembly factor Psb27 revealed variations in its lipid modification

2021 ◽  
Author(s):  
Jan Lambertz ◽  
Pasqual Liauw ◽  
Julian P. Whitelegge ◽  
Marc M. Nowaczyk
Keyword(s):  
Mass Spectrometry ◽  
Photosystem Ii ◽  
Protein Complexes ◽  
Exchange Chromatography ◽  
Mass Spectrometry Analysis ◽  
Large Network ◽  
Spectrometry Analysis ◽  
Assembly Factor ◽  
Membrane Protein Complexes ◽  
High Level

AbstractThe assembly of large, multi-cofactor membrane protein complexes like photosystem II (PSII) requires a high level of coordination. The process is facilitated by a large network of auxiliary proteins that bind transiently to unassembled subunits, preassembled modules or intermediate states of PSII, which are comprised of a subset of subunits. However, analysis of these immature, partially assembled PSII complexes is hampered by their low abundance and intrinsic instability. In this study, PSII was purified from the thermophilic cyanobacterium Thermosynechococcus elongatus via Twin-Strep-tagged CP43 and further separated by ion exchange chromatography into mature and immature complexes. Mass spectrometry analysis of the immature Psb27-PSII intermediate revealed six different Psb27 proteoforms with distinct lipid modifications. The maturation and functional role of thylakoid localized lipoproteins are discussed.

scholarly journals Getting more out of co-immunoprecipitation mass spectrometry experiments by reducing interference using FAIMS.

2021 ◽  
Author(s):  
Ching-Seng Ang ◽  
Joanna Sacharz ◽  
Michael G Leeming ◽  
Shuai Nie ◽  
Swati Varshney ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Ion Mobility ◽  
Protein Complexes ◽  
Affinity Purification ◽  
Mass Spectrometry Analysis ◽  
Modern Biology ◽  
Spectrometry Analysis ◽  
Affinity Enrichment ◽  
Unbiased Manner ◽  
Gas Phase Ion

Co-immunoprecipitation of proteins coupled to mass spectrometry has transformed modern biology understanding of protein interaction networks. These approaches exploit the selective isolation of tagged proteins by affinity enrichment / purification to identify protein binding partners at scale and in an unbiased manner. In instances where a suitable antibody is not be available it is common to graft synthetic tags such as FLAG or His Tags onto target protein sequences allowing the use of commercially available and validated antibodies for affinity purification. To allow the selective elution of protein complexes competitive displacement using a large molar excess of the tag peptide is widely used. Yet, this creates downstream challenges for the mass spectrometry analysis due to the presence of large quantities of a contaminating peptide. Here, we demonstrate that Field Asymmetric Ion Mobility Spectrometry (FAIMS), a gas phase ion separation device can be applied to FLAG-Tag and His-Tag pull down assay to increase the depth of protein coverage in these experiments. By excluding tag peptides based on their ion mobility profiles we demonstrate that single compensation voltage, or stepped compensation voltages strategies can significantly increase the coverage of total proteins by up to 2.5-fold and unique proteins by up to 15-fold versus experiments that do not use FAIMS. Combined these results highlight FAIMS is able to improve proteome depth by excluding interfering peptides without the need for additional sample handling or altering sample preparation protocols.

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 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.

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