Use of different proteases working in acidic conditions to improve sequence coverage and resolution in hydrogen/deuterium exchange of large proteins.
The combination of hydrogen exchange and mass spectrometry has been widely used in structural biology, providing views on protein structure and protein dynamics. One of the constraints is to use proteases working at low pH and low temperature to limit back-exchange during proteolysis. Although pepsin works in these conditions and is currently used in such experiments, sequence coverage is not always complete especially for large proteins, and the spatial resolution of the exchange rate is limited by the size of the resulting peptides. In this study we tried two other proteases, protease type XIII from Aspergillus saitoi and protease type XVIII from Rhizhopus species. The penicillin-binding protein X (PBP-2X*), a 77-kDa protein, was selected as a model. Like pepsin, neither of these proteases is really specific, but we found very good reproducibility in the digestion pattern. Compared with using pepsin alone, combining the results of the three independent proteolyses increased the coverage for the peptide mapping, thus avoiding missing some potentially interesting regions of the protein. Furthermore, we obtained a better spatial resolution for deuterium incorporation data, specifying accurately the deuterated regions.
Cravello
Laetitia
L
Laboratoire de Spectrométrie de Masse des Protéines, Institut de Biologie Structurale (UMR 5075 CEA/CNRS/UJF), 41 rue Jules Horowitz, F-38027 Grenoble Cedex 1, France.
Lascoux
David
D
Forest
Eric
E
D000595:Amino Acid Sequence; D001230:Aspergillus / Q000201:enzymology; D003903:Deuterium / Q000737:chemistry; D041961:Deuterium Exchange Measurement; D010450:Endopeptidases / Q000378:metabolism*; D006859:Hydrogen / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007553:Isotope Labeling; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010434:Pepsin A / Q000378:metabolism; D010449:Peptide Mapping; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D011506:Proteins; D006859:Hydrogen; D003903:Deuterium; D010450:Endopeptidases; D010434:Pepsin A