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Sulfate radical anion as a new reagent for fast photochemical oxidation of proteins.

ABSTRACT: The focus is to expand the original design of fast photochemical oxidation of proteins (FPOP) and introduce SO(4)(-•), generated by 248 nm homolysis of low millimolar levels of persulfate, as a radical reactant in protein footprinting. FPOP is a chemical approach to footprinting proteins and protein complexes by "snapshot" reaction with free radicals. The radical used until now is the OH radical, and it provides a measure of residue-resolved solvent accessibility of the native protein. We show that FPOP can accommodate other reagents, increasing its versatility. The new persulfate FPOP system is a potent, nonspecific, and tunable footprinting method; 3-5 times less persulfate is needed to give the same global levels of modification as seen with OH radicals. Although solvent-exposed His and Tyr residues are more reactive with SO(4)(-•) than with (•)OH, oxidation of apomyoglobin and calmodulin shows that (•)OH probes smaller accessible areas than SO(4)(-•), with the possible exception of histidine. His64, an axial ligand in the heme-binding pocket of apomyoglobin, is substantially up-labeled by SO(4)(-•) relative to (•)OH. Nevertheless, the kinds of modification and residue selectivity for both reagent radicals are strikingly similar. Thus, the choice of these reagents relies on the physical properties, particularly the membrane permeability, of the radical precursors.

SUBMITTER: Gau BC

PROVIDER: S-EPMC2939269 | biostudies-literature | 2010 Sep

REPOSITORIES: biostudies-literature

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Sulfate radical anion as a new reagent for fast photochemical oxidation of proteins.

Gau Brian C BC Chen Hao H Zhang Yun Y Gross Michael L ML

Analytical chemistry 20100901 18

The focus is to expand the original design of fast photochemical oxidation of proteins (FPOP) and introduce SO(4)(-•), generated by 248 nm homolysis of low millimolar levels of persulfate, as a radical reactant in protein footprinting. FPOP is a chemical approach to footprinting proteins and protein complexes by "snapshot" reaction with free radicals. The radical used until now is the OH radical, and it provides a measure of residue-resolved solvent accessibility of the native protein. We show t ...[more]

PMID: 20738105

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Project description:Fast Photochemical Oxidation of proteins (FPOP) coupled with mass spectrometry (MS) has become an invaluable tool in structural proteomics to interrogate protein interactions, structure, and protein conformational dynamics as a function of solvent accessibility. In recent years, the scope of FPOP, a hydroxyl radical protein foot printing (HRPF) technique, has been expanded to protein labeling in live cell cultures, providing the means to study protein interactions in the convoluted cellular environment. In-cell protein modifications can provide insight into ligand induced structural changes or conformational changes accompanying protein complex formation, all within the cellular context. Protein footprinting has been accomplished employing a customary flow-based system and a 248 nm KrF excimer laser to yield hydroxyl radicals via photolysis of hydrogen peroxide, requiring 20 minutes of analysis for one cell sample.To facilitate time-resolved FPOP experiments, the use of a new 6-well plate-based IC-FPOP platform was pioneered. In the current system, a single laser pulse irradiates one entire well, which truncates the FPOP experimental time frame resulting in 20 seconds of analysis time, a 60-fold decrease. This greatly reduced analysis time makes it possible to research cellular mechanisms such as biochemical signaling cascades, protein folding, and differential experiments (i.e., drug-free vs. drug bound) in a time-dependent manner. This new instrumentation, entitled Platform Incubator with Movable XY Stage (PIXY), allows the user to perform cell culture and IC-FPOP directly on the optical bench using a platform incubator with temperature, CO2 and humidity control. The platform also includes a positioning stage, peristaltic pumps, and mirror optics for laser beam guidance. IC-FPOP conditions such as optics configuration, flow rates, transient transfections, and H2O2 concentration in PIXY have been optimized and peer-reviewed. Automation of all components of the system will reduce human manipulation and increase throughput.

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Sulfate radical anion as a new reagent for fast photochemical oxidation of proteins.

Publications

Sulfate radical anion as a new reagent for fast photochemical oxidation of proteins.

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