Spatially resolved protein hydrogen exchange measured by matrix-assisted laser desorption ionization in-source decay
Research output: Contribution to journal › Journal article › Research › peer-review
Standard
Spatially resolved protein hydrogen exchange measured by matrix-assisted laser desorption ionization in-source decay. / Rand, Kasper Dyrberg; Bache, Nicolai; Nedertoft, Morten M; Jørgensen, Thomas.
In: Analytical Chemistry, Vol. 83, No. 23, 2011, p. 8859-62.Research output: Contribution to journal › Journal article › Research › peer-review
Harvard
APA
Vancouver
Author
Bibtex
}
RIS
TY - JOUR
T1 - Spatially resolved protein hydrogen exchange measured by matrix-assisted laser desorption ionization in-source decay
AU - Rand, Kasper Dyrberg
AU - Bache, Nicolai
AU - Nedertoft, Morten M
AU - Jørgensen, Thomas
PY - 2011
Y1 - 2011
N2 - 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(a) 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.
AB - 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(a) 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.
U2 - 10.1021/ac202468v
DO - 10.1021/ac202468v
M3 - Journal article
C2 - 22054221
VL - 83
SP - 8859
EP - 8862
JO - Industrial And Engineering Chemistry Analytical Edition
JF - Industrial And Engineering Chemistry Analytical Edition
SN - 0003-2700
IS - 23
ER -
ID: 40129308