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Fast Photochemical Oxidation of Proteins Coupled with Mass Spectrometry

[ Vol. 26 , Issue. 1 ]

Author(s):

Liuqing Shi and Michael L. Gross*   Pages 27 - 34 ( 8 )

Abstract:


Background: Determination of the composition and some structural features of macromolecules can be achieved by using structural proteomics approaches coupled with mass spectrometry (MS). One approach is hydroxyl radical protein footprinting whereby amino-acid side chains are modified with reactive reagents to modify irreversibly a protein side chain. The outcomes, when deciphered with mass-spectrometry-based proteomics, can increase our knowledge of structure, assembly, and conformational dynamics of macromolecules in solution. Generating the hydroxyl radicals by laser irradiation, Hambly and Gross developed the approach of Fast Photochemical Oxidation of Proteins (FPOP), which labels proteins on the sub millisecond time scale and provides, with MS analysis, deeper understanding of protein structure and protein-ligand and protein- protein interactions. This review highlights the fundamentals of FPOP and provides descriptions of hydroxyl-radical and other radical and carbene generation, of the hydroxyl labeling of proteins, and of determination of protein modification sites. We also summarize some recent applications of FPOP coupled with MS in protein footprinting.

Conclusion: We survey results that show the capability of FPOP for qualitatively measuring protein solvent accessibility on the residue level. To make these approaches more valuable, we describe recent method developments that increase FPOP’s quantitative capacity and increase the spatial protein sequence coverage. To improve FPOP further, several new labeling reagents including carbenes and other radicals have been developed. These growing improvements will allow oxidative- footprinting methods coupled with MS to play an increasingly significant role in determining the structure and dynamics of macromolecules and their assemblies.

Keywords:

Hydroxyl radical labeling, Fast Photochemical Oxidation of Proteins (FPOP), protein footprinting, liquid chromatography- mass spectrometry, solvent accessibility, protein high order structure, amyloids, protein folding, epitope mapping.

Affiliation:

Department of Chemistry, Washington University, St. Louis, MO 63130, Department of Chemistry, Washington University, St. Louis, MO 63130

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