Hydroxyl radical protein footprinting for analysis of higher order structure

Abstract

Tabled 1aAdapted from Johnson et al. [11].Hydrogen deuterium exchangeGlycine ethyl esterDiethylpyrocarbonateN-Ethylmaleimide (NEM)Fast photochemical oxidation of proteinsLabeling timescaleSeconds to hoursSeconds to minutesMinutesMinutes to hoursMicrosecondsType of labelReversibleReversibleReversible for most residuesIrreversible for LysIrreversibleIrreversibleSensitivity/specificityBackbone amide groupsCarboxylic acidsNucleophilic residuesN terminus, His, Lys, Tyr, Ser, Thr, and CysCys19 out of 20 amino acids (excluding Gly)Chemicals/materialsCommonly used buffers under physiological conditionsQuench solutionspH 2.5 (strong or weak acid)0°CDenaturing (urea,guanidine HCl)Reducing agents (TCEP)Pepsin digestionEDC stock solutionprepared freshly in PBS bufferMES/NaOH (pH = 5.5),PBS (pH = 6, 6.5, 7, 7.5),HEPES (pH = 8, 8.5)DEPC in acetonitrile in a molar excess of 4 10 mM imidazole added to quench the reactionNEM solution10 mm HEPES5% DMSOVarious buffer solutions based on proteinH2O2free glutamine radical scavengerQuench solution: free methionine and catalase (in vitro)LimitationsReversibility of the label limits its use for complex samples that require longer separation timesCarboxylic acid-specific labeling limits the proteins that can be studiedFree cysteine scramblingCysteine-specific labeling limits the proteins that can be studiedResidue-reactivity differences, complex data analysis, and expense of excimer lasera Adapted from Johnson et al. [11]. Open table in a new tab Hydroxyl radicals can be generated using various methods, including Fenton chemistry, radiolysis of water, and photolysis of hydrogen peroxide. The labeling timescale is based on the radical generation method with some labeling proteins on the nano- or microsecond timescale, which is faster than all other footprinting methods. HRPF has been successful in studying HOS in varied applications, including epitope mapping, ion channel dynamics, and viral protein studies. HRPF has also been used to study intact cells and in vivo systems.ADVANTAGES:General labeling strategy that can modify 19 of the 20 amino acids.Labeling is compatible with several widely used buffer systems.The irreversible nature of the label provides flexibility for postlabeling sample handling.Can identify protein interaction sites and regions of conformational changes.Can analyze complex systems such as cell lysates, intact cells, and Caenorhabditis elegans.Advancements in dosimetry have increased the quantitative power of the method.There have been efforts to automate labeling leading to higher throughput.CHALLENGES:Data analysis is complex, time consuming, and there is not a universal data analysis platform.Generating hydroxyl radicals on a fast timescale requires special instrumentation.Lower resolution than other structural methods.Difficult to identify interacting proteins in complex systems.Declaration of interestsL.M.J. discloses a significant financial interest in GenNext Technologies, Inc., an early-stage company seeking to commercialize technologies for protein higher-order structure analysis. Hydroxyl radicals can be generated using various methods, including Fenton chemistry, radiolysis of water, and photolysis of hydrogen peroxide. The labeling timescale is based on the radical generation method with some labeling proteins on the nano- or microsecond timescale, which is faster than all other footprinting methods. HRPF has been successful in studying HOS in varied applications, including epitope mapping, ion channel dynamics, and viral protein studies. HRPF has also been used to study intact cells and in vivo systems. ADVANTAGES:General labeling strategy that can modify 19 of the 20 amino acids.Labeling is compatible with several widely used buffer systems.The irreversible nature of the label provides flexibility for postlabeling sample handling.Can identify protein interaction sites and regions of conformational changes.Can analyze complex systems such as cell lysates, intact cells, and Caenorhabditis elegans.Advancements in dosimetry have increased the quantitative power of the method.There have been efforts to automate labeling leading to higher throughput. General labeling strategy that can modify 19 of the 20 amino acids. Labeling is compatible with several widely used buffer systems. The irreversible nature of the label provides flexibility for postlabeling sample handling. Can identify protein interaction sites and regions of conformational changes. Can analyze complex systems such as cell lysates, intact cells, and Caenorhabditis elegans. Advancements in dosimetry have increased the quantitative power of the method. There have been efforts to automate labeling leading to higher throughput. CHALLENGES:Data analysis is complex, time consuming, and there is not a universal data analysis platform.Generating hydroxyl radicals on a fast timescale requires special instrumentation.Lower resolution than other structural methods.Difficult to identify interacting proteins in complex systems. Data analysis is complex, time consuming, and there is not a universal data analysis platform. Generating hydroxyl radicals on a fast timescale requires special instrumentation. Lower resolution than other structural methods. Difficult to identify interacting proteins in complex systems. Declaration of interestsL.M.J. discloses a significant financial interest in GenNext Technologies, Inc., an early-stage company seeking to commercialize technologies for protein higher-order structure analysis. L.M.J. discloses a significant financial interest in GenNext Technologies, Inc., an early-stage company seeking to commercialize technologies for protein higher-order structure analysis.