Electrospray ionization mass spectrometry as a method for studying the high-pressure denaturation of proteins.
High-pressure denaturation of proteins can provide important information concerning their folding and function. These studies require expensive and complicated equipment. In this paper, we present a new convenient method for studying high-pressure denaturation of proteins combining DHX (deuterium-hydrogen exchange) and electrospray ionization MS. Application of various values of pressure causes different degrees of protein unfolding resulting in molecules with a different number of protons available for exchange with deuterons. After decompression a protein refolds and a certain number of deuterons are trapped within the hydrophobic core of a refolded protein. Redissolving the deuterated protein in an aqueous buffer initiates the DHX of amides located on the protein surface only, which can be monitored under atmospheric pressure by MS. Depending on the degree of deuteration after high-pressure treatment, the DHX kinetics are different and indicate how many deuterons were trapped in the protein after refolding. The dependence of this number on pressure gives information on the denaturation state of a protein. The distribution of deuterium along the sequence of a high-pressure-denatured protein was studied the ECD (electron-capture-induced dissociation) on a Fourier-transform mass spectrometer, enabling the monitoring of high-pressure denaturation with single amino acid resolution.
Stefanowicz
Piotr
P
Faculty of Chemistry, University of Wroclaw, 50-383 Wroclaw, Poland. stp@wchuwr.pl
Petry-Podgorska
Inga
I
Kowalewska
Karolina
K
Jaremko
Lukasz
L
Jaremko
Mariusz
M
Szewczuk
Zbigniew
Z
D000818:Animals; D002627:Chemistry, Physical / Q000379:methods; D003903:Deuterium / Q000737:chemistry; D041961:Deuterium Exchange Measurement; D006801:Humans; D006859:Hydrogen / Q000737:chemistry; D007768:Lactalbumin / Q000737:chemistry; D009113:Muramidase / Q000737:chemistry; D011312:Pressure; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D017550:Spectroscopy, Fourier Transform Infrared; D013816:Thermodynamics; D025801:Ubiquitin / Q000737:chemistry
D025801:Ubiquitin; D006859:Hydrogen; D007768:Lactalbumin; D003903:Deuterium; D009113:Muramidase