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Folding and assembly of large macromolecular complexes monitored by hydrogen-deuterium exchange and mass spectrometry.

Suchanova B, Tuma R - Microb. Cell Fact. (2008)

Bottom Line: HDX-MS became a valuable tool to follow protein folding, assembly and aggregation.The methodology has a wide range of applications in biotechnology ranging from quality control for over-expressed proteins and their complexes to screening of potential ligands and inhibitors.This review provides an introduction to protein folding and assembly followed by the principles of HDX and MS detection, and concludes with selected examples of applications that might be of interest to the biotechnology community.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Biotechnology, University of Helsinki, Finland. r.tuma@leeds.ac.uk.

ABSTRACT
Recent advances in protein mass spectrometry (MS) have enabled determinations of hydrogen deuterium exchange (HDX) in large macromolecular complexes. HDX-MS became a valuable tool to follow protein folding, assembly and aggregation. The methodology has a wide range of applications in biotechnology ranging from quality control for over-expressed proteins and their complexes to screening of potential ligands and inhibitors. This review provides an introduction to protein folding and assembly followed by the principles of HDX and MS detection, and concludes with selected examples of applications that might be of interest to the biotechnology community.

No MeSH data available.


Schematics of on-line folding and pulse labeling apparatus (based on [102]).
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Figure 5: Schematics of on-line folding and pulse labeling apparatus (based on [102]).

Mentions: Pulse-labeling HDX-MS techniques enable detection and characterization of transient folding intermediates which are not readily populated under equilibrium conditions [101]. An on-line HDX pulse-labeling ESI MS apparatus was developed and tested on myoglobin folding and heme incorporation [86,102] (Fig. 5). The method is based on tandem mixing chambers: the first one serves to initiate folding/assembly reaction while the second one is used to stop the folding reaction and transiently expose the products to deuterium label. Subsequently, the exchange is quenched and the incorporated label is quantified by MS. This provides a "snapshot" of protection at a particular stage of the reaction. Usually, this is applied to intact proteins but there is no principal obstacle in carrying out pepsin digestion and LC-MS analysis and obtain region-specific exchange kinetics.


Folding and assembly of large macromolecular complexes monitored by hydrogen-deuterium exchange and mass spectrometry.

Suchanova B, Tuma R - Microb. Cell Fact. (2008)

Schematics of on-line folding and pulse labeling apparatus (based on [102]).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2365927&req=5

Figure 5: Schematics of on-line folding and pulse labeling apparatus (based on [102]).
Mentions: Pulse-labeling HDX-MS techniques enable detection and characterization of transient folding intermediates which are not readily populated under equilibrium conditions [101]. An on-line HDX pulse-labeling ESI MS apparatus was developed and tested on myoglobin folding and heme incorporation [86,102] (Fig. 5). The method is based on tandem mixing chambers: the first one serves to initiate folding/assembly reaction while the second one is used to stop the folding reaction and transiently expose the products to deuterium label. Subsequently, the exchange is quenched and the incorporated label is quantified by MS. This provides a "snapshot" of protection at a particular stage of the reaction. Usually, this is applied to intact proteins but there is no principal obstacle in carrying out pepsin digestion and LC-MS analysis and obtain region-specific exchange kinetics.

Bottom Line: HDX-MS became a valuable tool to follow protein folding, assembly and aggregation.The methodology has a wide range of applications in biotechnology ranging from quality control for over-expressed proteins and their complexes to screening of potential ligands and inhibitors.This review provides an introduction to protein folding and assembly followed by the principles of HDX and MS detection, and concludes with selected examples of applications that might be of interest to the biotechnology community.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Biotechnology, University of Helsinki, Finland. r.tuma@leeds.ac.uk.

ABSTRACT
Recent advances in protein mass spectrometry (MS) have enabled determinations of hydrogen deuterium exchange (HDX) in large macromolecular complexes. HDX-MS became a valuable tool to follow protein folding, assembly and aggregation. The methodology has a wide range of applications in biotechnology ranging from quality control for over-expressed proteins and their complexes to screening of potential ligands and inhibitors. This review provides an introduction to protein folding and assembly followed by the principles of HDX and MS detection, and concludes with selected examples of applications that might be of interest to the biotechnology community.

No MeSH data available.