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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.


Example of HDX detected by mass spectra. m/z isotopic envelopes for EX2 (A) and EX1 (B) limit of exchange. Data obtained for a region that is situated within the subunit interface in ϕ8 P4 hexamer [114].
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Figure 4: Example of HDX detected by mass spectra. m/z isotopic envelopes for EX2 (A) and EX1 (B) limit of exchange. Data obtained for a region that is situated within the subunit interface in ϕ8 P4 hexamer [114].

Mentions: Digestion with pepsin or other acidic proteases is rather non-specific and under given conditions (e.g. digestion time, denaturant concentration, protease-to-protein ratio) yields overlapping fragments with lengths ranging from 5 to 15 amino acids. The same set of peptide fragments is obtained for a given protein and digestion conditions [75]. This assures that consistent and reproducible data sets are obtained in independent runs. Each of the fragments acquires one or more positive charges during ionization and produces a typical isotopic envelope in the mass spectrum (Fig 4A). The relative abundance of different ionization states and fragments strongly depends on the particular ionization interface and mass spectrometer design. Hence, it is advisable to use one MS instrument throughout the whole study.


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

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

Example of HDX detected by mass spectra. m/z isotopic envelopes for EX2 (A) and EX1 (B) limit of exchange. Data obtained for a region that is situated within the subunit interface in ϕ8 P4 hexamer [114].
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Example of HDX detected by mass spectra. m/z isotopic envelopes for EX2 (A) and EX1 (B) limit of exchange. Data obtained for a region that is situated within the subunit interface in ϕ8 P4 hexamer [114].
Mentions: Digestion with pepsin or other acidic proteases is rather non-specific and under given conditions (e.g. digestion time, denaturant concentration, protease-to-protein ratio) yields overlapping fragments with lengths ranging from 5 to 15 amino acids. The same set of peptide fragments is obtained for a given protein and digestion conditions [75]. This assures that consistent and reproducible data sets are obtained in independent runs. Each of the fragments acquires one or more positive charges during ionization and produces a typical isotopic envelope in the mass spectrum (Fig 4A). The relative abundance of different ionization states and fragments strongly depends on the particular ionization interface and mass spectrometer design. Hence, it is advisable to use one MS instrument throughout the whole study.

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.