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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.


Practical aspects of HDX measurement. (A) Simplified exchange and protease digestion protocol. (B) Cooled LC-MS setup, INJ = injector [64].
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Figure 3: Practical aspects of HDX measurement. (A) Simplified exchange and protease digestion protocol. (B) Cooled LC-MS setup, INJ = injector [64].

Mentions: The extent and rate of HDX is measured from mass increases of peptide fragments after enzymatic cleavage of a protein (Fig. 3). The exchange is usually initiated by diluting the protein into D2O exchange buffer (Fig. 3A). The sample is incubated under the exchange conditions for the desired exchange period (usually 30 s to 10 h) and then quenched by rapid acidification to pH 2.5 on ice (usually done with formic acid). This effectively slows down any further exchange (so called exchange-in) and minimizes pickup of hydrogens during subsequent handling in H2O solutions (so called back-exchange). The quenching solution may be supplemented with a denaturant (e.g. guanidine hydrochloride or urea) to facilitate dissociation of stable complexes prior to proteolytic digestion. The quenched sample may be analyzed immediately or flash-frozen and stored for up to several weeks in liquid nitrogen for off-line analysis.


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

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

Practical aspects of HDX measurement. (A) Simplified exchange and protease digestion protocol. (B) Cooled LC-MS setup, INJ = injector [64].
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Practical aspects of HDX measurement. (A) Simplified exchange and protease digestion protocol. (B) Cooled LC-MS setup, INJ = injector [64].
Mentions: The extent and rate of HDX is measured from mass increases of peptide fragments after enzymatic cleavage of a protein (Fig. 3). The exchange is usually initiated by diluting the protein into D2O exchange buffer (Fig. 3A). The sample is incubated under the exchange conditions for the desired exchange period (usually 30 s to 10 h) and then quenched by rapid acidification to pH 2.5 on ice (usually done with formic acid). This effectively slows down any further exchange (so called exchange-in) and minimizes pickup of hydrogens during subsequent handling in H2O solutions (so called back-exchange). The quenching solution may be supplemented with a denaturant (e.g. guanidine hydrochloride or urea) to facilitate dissociation of stable complexes prior to proteolytic digestion. The quenched sample may be analyzed immediately or flash-frozen and stored for up to several weeks in liquid nitrogen for off-line analysis.

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.