Using stable-isotope-labeled proteins for hydrogen exchange studies in complex mixtures.
The use of mass spectrometry to measure hydrogen exchange rates for individual proteins in complex mixtures is described. Incorporation of stable-isotope-labeled (SIL) amino acids into a protein of interest during overexpression in bacteria produced distinctive isotope patterns in mass spectra of peptic peptides from the labeled protein. The isotope pattern was used as a signature for peptides originating from the SIL protein. In addition, stable-isotope labeling simplified identification of the peptic peptides by providing partial amino acid composition information. Despite the complex isotope patterns associated with SIL peptides, hydrogen exchange rates could still be measured for peptides from SIL protein and were found to be the same as exchange rates for unlabeled protein. Hydrogen exchange in a single protein of interest was measured in a complex mixture of proteins, a bacterial cell lysate. This methodology, which includes easy recognition of peptic peptides from the protein(s) of interest during hydrogen exchange studies in heterogeneous systems, will permit analysis of structural properties and dynamics of large protein complexes and complex protein systems.
Engen
John R
JR
C-ACS, Chemistry Division, Los Alamos National Laboratory, New Mexico 87545, USA.
Bradbury
E Morton
EM
Chen
Xian
X
D003600:Cytosol / Q000737:chemistry; D003903:Deuterium / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry; D006859:Hydrogen / Q000378:metabolism*; D007554:Isotopes / Q000378:metabolism; D046911:Macromolecular Substances; D013058:Mass Spectrometry / Q000379:methods; D011485:Protein Binding; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*
D007554:Isotopes; D046911:Macromolecular Substances; D011506:Proteins; D006859:Hydrogen; D003903:Deuterium