Implementing fast photochemical oxidation of proteins (FPOP) as a footprinting approach to solve diverse problems in structural biology

Zhang, Bojie; Cheng, Ming; Rempel, Don L.; Gross, Michael L.

doi:10.1016/j.ymeth.2018.05.016

Cited by 36 publications

(37 citation statements)

References 58 publications

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“…The area of incidence between the laser light and the sample is considerably larger for microtiter single pulse photolysis. The use of cylindrical lenses to asymmetrically compress the beam to the shape of the capillary allows traditional FPOP to reach higher laser fluences at more modest pulse energies [27] . Since the area illuminated is much larger in the microtiter plate, the laser energy per pulse required is considerably higher, potentially requiring a higher power laser.…”

Section: Resultsmentioning

confidence: 99%

Towards high-throughput fast photochemical oxidation of proteins: Quantifying exposure in high fluence microtiter plate photolysis

Riaz

Misra

Sharp

2018

Analytical Biochemistry

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Protein structural analysis by mass spectrometry has gained significant popularity in recent years, including high-resolution protein topographical mapping by fast photochemical oxidation of proteins (FPOP). The ability to provide protein topographical information at moderate spatial resolution makes FPOP an attractive technology for the protein pharmaceutical discovery and development processes. However, current technology limits the throughput and requires significant manual sample manipulation. Similarly, as FPOP is being used on larger samples, sample flow through the capillary becomes challenging. No systematic comparison of the performance of static flash photolysis with traditional flow FPOP has been reported. Here, we evaluate a 96-well microtiter-based laser flash photolysis method for the topographical probing of proteins, which subsequently could be used to analyze higher order structure of the protein in a high-throughput fashion with minimal manual sample manipulation. We used multiple metrics to compare microtiter FPOP performance with that of traditional flow FPOP: adenine-based hydroxyl radical dosimetry, oxidation efficiency of a model peptide, and hydroxyl radical protein footprint of myoglobin. In all cases, microtiter plate FPOP performed comparably with traditional flow FPOP, requiring a small fraction of the time for exposure. This greatly reduced sample exposure time, coupled with automated sample handling in 96-well microtiter plates, makes microtiter-based FPOP an important step in achieving the throughput required to adapt hydroxyl radical protein footprinting for screening purposes.

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Section: Resultsmentioning

confidence: 99%

Towards high-throughput fast photochemical oxidation of proteins: Quantifying exposure in high fluence microtiter plate photolysis

Riaz

Misra

Sharp

2018

Analytical Biochemistry

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“…We demonstrated that this normalization allows us to obtain identical footprints for proteins in widely differing scavenging backgrounds, which is key for comparability studies. Incorporation of a pinhole aperture as previously reported 36 allows for investigators to change fluence (and, therefore, the amount of radicals generated) without need of correcting for altered beam area by changing the flow rate.…”

Section: Resultsmentioning

confidence: 99%

“…buffer, ligand, detergent, lipids, excipients, allosteric modulator, etc.) 23,36 and isotope dilution GC-MS 37 . Perhaps the simplest was to replace a portion of the glutamine radical scavenger with adenine, which has an almost identical rate of reaction as glutamine 38 .…”

Section: Introductionmentioning

confidence: 99%

“…Xie and Sharp previously showed that monitoring the UV absorbance of the adenine in an FPOP reaction after quenching measures the effective hydroxyl radical concentration, and allows the researcher to ensure that two samples experienced equivalent FPOP conditions for comparison 39 . Regardless of the method, post-quenching radical dosimetry is quickly becoming a standard part of best practices for any FPOP experimental protocol 36 .…”

Section: Introductionmentioning

confidence: 99%

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Real Time Normalization of Fast Photochemical Oxidation of Proteins Experiments by Inline Adenine Radical Dosimetry

Sharp

Misra

Egan³

et al. 2018

Preprint

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Hydroxyl radical protein footprinting (HRPF) is a powerful method for measuring protein topography, allowing researchers to monitor events that alter the solvent accessible surface of a protein (e.g. ligand binding, aggregation, conformational changes, etc.) by measuring changes in the apparent rate of reaction of portions of the protein to hydroxyl radicals diffusing in solution. Fast Photochemical Oxidation of Proteins (FPOP) offers an ultra-fast benchtop method for performing HRPF, photolyzing hydrogen peroxide using a UV laser to generate high concentrations of hydroxyl radicals that are consumed on roughly a microsecond timescale. The broad reactivity of hydroxyl radicals means that almost anything added to the solution (e.g. ligands, buffers, excipients, etc.) will scavenge hydroxyl radicals, altering their half-life and changing the effective radical concentration experienced by the protein. Similarly, minute changes in peroxide concentration, laser fluence, and buffer composition can alter the effective radical concentration, making reproduction of data challenging. Here, we present a simple method for radical dosimetry that can be carried out as part of the FPOP workflow, allowing for measurement of effective radical concentration in real time. Additionally, by modulating the amount of radical generated, we demonstrate that FPOP HRPF experiments carried out in buffers with widely differing levels of hydroxyl radical scavenging capacity can be normalized on the fly, yielding statistically indistinguishable results for the same conformer. This method represents a major step in transforming FPOP into a robust and reproducible technology capable of probing protein structure in a wide variety of contexts.

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“…Fast photochemical oxidation of proteins (FPOP), a method that generates hydroxyl radicals by UV laser-induced photolysis of hydrogen peroxide, has demonstrated the ability to react with a wide variety of amino acids on the surface of the proteins and label them without altering the native protein structure during labeling [7][8][9]. FPOP coupled with MS has been rapidly adopted to protein structural biology research in recent decades [7,[10][11][12]. However, FPOP coupled with MS for membrane protein analysis remains limited, in part due to the low extent of labeling that has been reported.…”

Section: Introductionmentioning

confidence: 99%

Self-Organized Amphiphiles are Poor Hydroxyl Radical Scavengers in Fast Photochemical Oxidation of Proteins Experiments

Cheng

Mobley²,

Misra³

et al. 2020

Preprint

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The analysis of membrane protein topography using fast photochemical oxidation of protein (FPOP) has been reported in recent years, but still underrepresented in literature. Based on the hydroxyl radical reactivity of lipids and other amphiphiles, it is believed that the membrane environment acts as a hydroxyl radical scavenger decreasing effective hydroxyl radical doses and resulting in less observed oxidation of proteins. Here, we investigated the effect of bulk hydroxyl radical scavenging in FPOP using both isolated cellular membranes as well as detergent micelles. We found no significant change in radical scavenging activity upon the addition of disrupted cellular membranes with the membrane concentration in the range of 0-25600 cell/μL using an inline radical dosimeter. We confirmed the non-scavenging nature of the membrane with the FPOP results of a soluble model protein in the presence of cell membranes, which showed no significant difference in oxidation with or without membranes. The use of detergents revealed that, while soluble detergent below the critical micelle concentration acts as a potent hydroxyl radical scavenger as expected, additional detergent has little to no hydroxyl radical scavenging effect once the critical micelle concentration is reached. These results suggest that any scavenging effect of membranes or organized amphiphilic membrane mimetics in FPOP experiments are not due to bulk hydroxyl radical scavenging, but may be due to a localized scavenging phenomenon.

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Implementing fast photochemical oxidation of proteins (FPOP) as a footprinting approach to solve diverse problems in structural biology

Cited by 36 publications

References 58 publications

Towards high-throughput fast photochemical oxidation of proteins: Quantifying exposure in high fluence microtiter plate photolysis

Towards high-throughput fast photochemical oxidation of proteins: Quantifying exposure in high fluence microtiter plate photolysis

Real Time Normalization of Fast Photochemical Oxidation of Proteins Experiments by Inline Adenine Radical Dosimetry

Self-Organized Amphiphiles are Poor Hydroxyl Radical Scavengers in Fast Photochemical Oxidation of Proteins Experiments

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