title: Spatially resolved protein hydrogen exchange measured by matrix-assisted laser desorption ionization in-source decay.
abstract: Mass spectrometry has become a powerful tool for measuring protein hydrogen exchange and thereby reveal the structural dynamics of proteins in solution. Here we describe the successful application of a matrix-assisted laser desorption ionization (MALDI) mass spectrometry approach based on in-source decay (ISD) to measure spatially resolved amide backbone hydrogen exchange. By irradiating deuterated protein molecules in a crystalline matrix with a high laser fluence, they undergo prompt fragmentation. Spatially resolved deuteration levels are readily obtained by mass analysis of consecutive fragment ions. MALDI ISD analysis of deuterated cytochrome c yielded an extensive series of c-fragment ions which originate from cleavage of nearly all N-C(α) bonds (Cys17 to Glu104) allowing for a detailed analysis of the deuterium content of the backbone amides. While hydrogen scrambling can be major concern when using mass spectrometric fragmentation to obtain detailed information on protein hydrogen exchange, we show that the level of hydrogen scrambling in our MALDI ISD measurements is negligible and that the known dynamic behavior of cytochrome c in solution is accurately reflected in the deuterium contents of the fragment ions. The developed method combines several attractive features from a practical point of view as it is simple to perform and it readily provides a detailed mapping of the dynamic structure of a protein in solution.
authors: Kasper D
authors: KD
authors: Rand
authors: Nicolai
authors: N
authors: Bache
authors: Morten M
authors: MM
authors: Nedertoft
authors: Thomas J D
authors: TJ
authors: Jørgensen
mesh_terms: D045304:Cytochromes c / Q000737:chemistry*; D003903:Deuterium / Q000737:chemistry; D006859:Hydrogen / Q000737:chemistry*; D017434:Protein Structure, Tertiary; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D006859:Hydrogen; D045304:Cytochromes c; D003903:Deuterium