title: Hydrogen exchange monitored by MALDI-TOF mass spectrometry for rapid characterization of the stability and conformation of proteins.
abstract: Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been used to monitor hydrogen exchange on entire proteins. Two alternative methods have been used to carry out the hydrogen exchange studies, exchanging deuteron (H to D experiments) or proton (D to H experiments). In the former case, the use of a deuterated matrix has made possible to overcome back-exchange problems and attain reproducible results. The methods presented have been used to determine the slow exchange core of the potato carboxypeptidase inhibitor in different folding states, and to differentially 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 exchange 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 minimal amounts of sample.
authors.affiliation: Institut de Biologia Fonamental and Departament de Bioquímica, Universitat Autònoma de Barcelona, 08193, Bellaterra, Spain.
authors.forename: J
authors.initials: J
authors.lastname: Villanueva
authors.forename: F
authors.initials: F
authors.lastname: Canals
authors.forename: V
authors.initials: V
authors.lastname: Villegas
authors.forename: E
authors.initials: E
authors.lastname: Querol
authors.forename: F X
authors.initials: FX
authors.lastname: Avilés
mesh_terms: D002268:Carboxypeptidases / Q000737:chemistry* / Q000235:genetics; D043422:Carboxypeptidases A; D020134:Catalytic Domain; D003903:Deuterium; D004792:Enzyme Precursors / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D006859:Hydrogen; D016297:Mutagenesis, Site-Directed; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics; D011480:Protease Inhibitors; D011487:Protein Conformation; D017510:Protein Folding; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D004792:Enzyme Precursors; D010940:Plant Proteins; D011480:Protease Inhibitors; D006859:Hydrogen; D003903:Deuterium; D002268:Carboxypeptidases; D043422:Carboxypeptidases A