Hydrogen exchange monitored by MALDI-TOF mass spectrometry for rapid characterization of the stability and conformation of proteins

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometr y (MALDI-TOF MS) has been used to monitor hydrogen exchange on entire prote ins. Two alternative methods have been used to carry out the hydrogen excha nge studies, exchanging deuteron (H to D experiments) or proton (D to H exp eriments). In the former case, the use of a deuterated matrix has made poss ible to overcome back-exchange problems and attain reproducible results. Th e methods presented ha,le been used to determine the slow exchange core of the potato carboxypeptidase inhibitor in different folding states, and to d ifferentially compare the activation domain of human procarboxypeptidase A2 versus three site-directed mutants of different conformational stability. In this work, we show that by using MALDI-TOF MS to monitor hydrogen exchan ge in entire proteins, it is possible to rapidly check the folding state of a protein and characterize mutational effects on protein conformation and stability, while requiring minima) amounts of sample. (C) 2000 Federation o f European Biochemical Societies.