Solid state synthesis hydroxyl radicals for high throughput structure determination of proteins and nucleic acids by oxidative footprinting
View Patent ↗The present invention generally relates to an apparatus for structural mapping of a macromolecule comprising an amount of persulfide effective to generate hydroxyl radicals upon contact with an aqueous solution. The present invention further relates to methods for structural mapping a macromolecule in an aqueous solution and methods for structural mapping a plurality of macromolecules in parallel, wherein each macromolecule is in a separate aqueous solution.
1. A method for structural mapping a macromolecule in an aqueous solution comprising: (a) contacting the aqueous solution containing the macromolecule with a persulfide of a persulfide-lined chamber such that hydroxyl radicals are generated; (b) incubating the aqueous solution containing the macromolecule such that the macromolecule is (i) cleaved by the hydroxyl radicals, thereby producing cleaved macromolecule fragments or (ii) oxidized by the hydroxyl radicals, thereby producing macromolecules of altered molecular weight; and (c) analyzing the cleaved macromolecule fragments or macromolecules of altered molecular weight produced in step (b) via a structural genomics method, thereby structural mapping the macromolecule.
2. The method of claim 1 , wherein the macromolecule is a protein, a DNA, an RNA, a protein-protein complex, a protein-DNA complex, a protein-RNA complex, or an RNA-DNA complex.
3. The method of claim 1 , wherein the structural genomics method is capillary automated sequencing analysis or mass spectrometry.
4. The method of claim 3 , wherein the mass spectrometry is matrix assisted laser desorption and ionization mass spectrometry.
5. The method of claim 1 , wherein the persulfide is contained in an apparatus for structural mapping of a macromolecule comprising an amount of persulfide effective to generate hydroxyl radicals upon contact with an aqueous solution.
6. The method of claim 1 , further comprising structural mapping a plurality of macromolecules in parallel, wherein each macromolecule is in a separate aqueous solution, wherein step (a) comprises contacting each of the plurality of the aqueous solutions with separate persulfide-lined chambers such that hydroxyl radicals are generated.
7. The method of claim 1 , wherein the persulfide-lined chambers are contained in an apparatus for structural mapping of a macromolecule comprising an amount of persulfide effective to generate hydroxyl radicals upon contact with an aqueous solution, wherein the apparatus comprises a microfabricated mixing device, wherein the microfabricated mixing device comprises a microfabricated chip, wherein the microfabricated chip comprises two or more persulfide-lined chambers for the structural mapping of two or more macromolecules.