This protocol presents a method to use inline radical dosimetry and a plasma light source to perform flash oxidation protein footprinting. This method replaces the hazardous UV laser to simplify and improve the reproducibility of fast photochemical oxidation of protein studies.
Hydroxyl Radical Protein Footprinting (HRPF) is an emerging and promising higher order structural analysis technique that provides information on changes in protein structure, protein-protein interactions, or protein-ligand interactions. HRPF utilizes hydroxyl radicals (▪OH) to irreversibly label a protein’s solvent accessible surface. The inherent complexity, cost, and hazardous nature of performing HRPF have substantially limited broad-based adoption in biopharma. These factors include: 1) the use of complicated, dangerous, and expensive lasers that demand substantial safety precautions; and 2) the irreproducibility of HRPF caused by background scavenging of ▪OH that limit comparative studies. This publication provides a protocol for operation of a laser-free HRPF system. This laser-free HRPF system utilizes a high energy, high-pressure plasma light source flash oxidation technology with in-line radical dosimetry. The plasma light source is safer, easier to use, and more efficient in generating hydroxyl radicals than laser-based HRPF systems, and the in-line radical dosimeter increases the reproducibility of studies. Combined, the laser-free HRPF system addresses and surmounts the mentioned shortcomings and limitations of laser-based techniques.
Protein conformation and associated higher order structure (HOS) are the principal determinants of proper biological function and aberrant behavior1. The same applies to biopharmaceuticals, whose structure and functional activity is dependent on various aspects of their production and environment. Biopharmaceutical change in HOS have been linked to adverse drug reactions (ADR) attributed to undesirable pharmacology and patient immunological response2,3. The appearance of ADRs has alerted the biopharmaceutical industry to the critical role that protein HOS plays in the safety and efficacy of biotherapeutics, and they have established the need for new and improved HOS analytics4.
Hydroxyl Radical Protein Footprinting (HRPF) is a promising technique to track the change in protein HOS. HRPF involves the irreversible labeling of a protein’s exterior with ▪OH followed with mass spectrometry (MS) analysis to identify the solvent accessible surface of the protein5,6,7. HRPF has successfully been used to detect defects in protein HOS and its function8,9, characterize the HOS of monoclonal antibodies (mAb)10,11,12,13, determine the binding Kd of a ligand14, and much more15,16,17,18,19. A common method to generate the ▪OH for HRPF is Fast Photochemical Oxidation of Proteins (FPOP), which employs high-energy, fast UV lasers to produce ▪OH from photolysis of H2O2. For the most part, FPOP uses expensive excimer lasers employing hazardous gas (KrF) that demands substantial safeguards to avoid respiratory and eye injury20. To avoid inhalation hazards, others have used frequency quadrupled neodymium yttrium aluminum garnet (Nd:YAG) lasers21, which eliminates the use of toxic gas but are still costly, require significant operational expertise, and demand extensive stray light controls to protect users from eye injury.
Although ample information can be obtained using HRPF, broad adoption in biopharma has not been met. Two barriers for the limited HRPF adoption include: 1) the use of dangerous and expensive lasers that demand substantial safety precautions20; and 2) the irreproducibility of HRPF caused by background scavenging of ▪OH that limit comparative studies22. To supplant laser use, a high-speed, high energy plasma flash photolysis unit was developed to safely perform FPOP in a facile manner. To improve on the irreproducibility of HRPF experiments, real-time radical dosimetry is implemented.
The practice of HRPF has been limited by irreproducibility attributed to background scavenging of ▪OH22. While ▪OH are excellent probes of protein topography, they also react with many constituents found in preparations, making it necessary to measure the effective concentration of radical available to oxidize a target protein. Variations in buffer preparation, hydrogen peroxide concentration, ligand properties, or photolysis can result in oxidation differences between control and experimental groups that create ambiguity in HOS differential studies. The addition of real-time radical dosimetry enables the adjustment of the effect ▪OH load and therefore increases the confidence and reproducibility during an HRPF experiment. The use of radical dosimetry in FPOP has been described elsewhere23,24,25, and is further discussed in detail in a recent publication26. Here, we describe the use of a novel flash photolysis system and real-time dosimetry to label equine apo-myoglobin (aMb), comparing levels of peptide oxidation in an FPOP experiment to that of obtained when using an excimer laser.
1. Installing the capillary tube
2. Installing an injection loop
3. Initialize the photolysis system
4. Determine actual ▪OH yield to test radical scavenging effects from the buffer.
5. Modification of protein to detect changes in higher order structure.
6. Shut the system down
7. Sample preparation and liquid chromatography-mass spectrometry
8. For a differential study, repeat steps 5-7 on the second condition.
NOTE: To confirm reproducibility, two biological replicates in addition to technical triplicates for each condition are recommended.
The high-pressure plasma source coupled with real-time dosimetry allows better control of ▪OH yield to observe changes in higher-order protein structure more accurately. The addition of adenine allows for an effective real-time radical dosimeter. Upon oxidation, adenine loses UV absorbance at 265 nm (Figure 2A). The change in adenine absorbance is directly related to the concentration of radicals available for HRPF thus providing a means to effectively monitor changes in radical concentration in the presence of radical scavengers like buffers, excipients, and ligands (Figure 2B).
Apomyoglobin (aMb) was modified in the presence of 100 mM H2O2 and 1 mM adenine at four increasing plasma voltages (Figure 3). Six peptides were detected with a linear increase in oxidation with the change of adenine UV 265 nm absorbance. Adenine UV absorbance is a surrogate for effective ▪OH concentration. The linear change in oxidation versus the change in radical concentration confirms the absence of artifactual change in the protein’s higher-order structure during labeling. Furthermore, the extent of oxidation detected with the high-pressure plasma source was much higher than the laser-based method. This increase arises from the high-pressure plasma source’s broad-spectrum UV emission spectrum (Figure 4). By producing a broad UV spectrum, the plasma source better overlaps the absorbance domain of H2O2 providing more efficient production of ▪OH through the photolysis of H2O2 (Figure 5). This is in direct comparison to the KrF Excimer laser and Nd:YAG laser, which are common sources that photolyze H2O2 in HRPF studies21,27,28. These lasers have a very narrow bandwidth at the lower end of H2O2’s photometric absorbance range. In comparison to the KrF excimer laser, the high pressure plasma source significantly increases the production of ▪OHs while concomitantly significantly decreasing the demand of required optical fluence (Figure 6).
Figure 1: Components of the flash photolysis platform. (A) Assembled capillary tube, union tube, nut and ferrule. The tip of the “lower end” of the capillary extends barely beyond the union tube. (B) Knurled nut on the product collector to insert the end of the capillary. (C) Labeled six-port injection valve. (D) Syringe pump with the syringe. Please click here to view a larger version of this figure.
Figure 2: Change in adenine absorbance used for dosimetry. (A) Upon oxidation, adenine decreases in UV absorbance at 265 nm. (B) Change in adenine absorbance at 265 nm at different flash voltages for two buffers. Buffer 2 contains a radical scavenger which will decrease the change in adenine absorbance compared to buffer 1. To overcome the radical scavenging effects from buffer 2, increased flash voltage is required. Please click here to view a larger version of this figure.
Figure 3: Apomyoglobin dose response curve. (A) Average oxidation of six peptides vs adenine change in absorbance is shown. FPOP oxidation levels are shown in the dashed lines. (B) The six oxidized peptides are labeled on a crystal structure of myoglobin (PDB: 3RGK). Please click here to view a larger version of this figure.
Figure 4: Spectral Irradiance of the plasma lamp. The high-pressure Xe gas lamp emits broad spectrum UV radiation from 200-300 nm along with visible light. Please click here to view a larger version of this figure.
Figure 5: H2O2 photometric absorbance spectra. The black trace is the UV absorbance spectrum of H2O229. Highlighted in purple is the narrow wavelengths of the KrF excimer laser (248 nm) and Nd:YAG laser (265 nm) produces while the blue arrow represents the broad-spectrum plasma source which extends the range of useful H2O2 absorbance. Please click here to view a larger version of this figure.
Figure 6: Efficacy of the plasma source to photolyze H2O2. The change in adenine absorbance over increased plasma fluence demonstrates the concentration of ▪OHs generated. Highlighted in purple is a typical max change in absorbance produced from a KrF excimer laser25. Please click here to view a larger version of this figure.
There are several critical steps to ensure proper labeling of proteins during any HRPF experiment. First, an appropriate flow rate and source flash rate are selected to make certain each bolus of the sample is irradiated once. This ensures that the protein is exposed to a single bolus of newly formed ▪OH. Once a protein is oxidized, the higher order protein structure can be altered. To be confident the native protein structure is probed, each protein molecule must be modified in a single instant. Dosimetry can be used to test if the native protein structure is probed. As the concentration of hydroxyl radical increases the extent of oxidation linearly increases if the native protein structure is probed. However, if a large concentration of hydroxyl radicals causes the protein to partially unfold during labeling, then the extent of oxidation will significantly increase thus providing an effective means to ensure the native protein structure is probed.
It is also critical to ensure the reaction is quenched appropriately. This includes incorporating a proper radical scavenger mixed with the protein during the labeling process. For this method, adenine is not only used for dosimetry but also scavenges ▪OH limiting the lifetime of the radical as it labels the protein. By limiting the lifetime of the radical, confidence that the native protein structure is being modified is gained. Following the labeling process, it is important to collect the sample in a quench solution containing catalase and methionine amide which will break down excess H2O2 and scavenge ▪OH. Limiting the protein’s exposure to H2O2 ensures H2O2 induced oxidation is minimized.
It is also critical to appropriately select a protease for peptide digestion. Many enzymes cleave the protein at specific locations. For example, trypsin cleaves on the carboxyl side of arginine and lysine residues. This allows for simple data analysis, but if the protein of interest has a limited number of arginines and lysines, an alternative protease or a mixture of proteases might be required for acceptable peptide coverage during bottom-up proteomics. It is also advised to digest the modified protein before long term storage to avoid loss of oxidized protein due to aggregation and/or solubility issues.
Using HRPF to study changes in protein structure and protein interactions has been very successful11,30,31,32,33, but there are added complications while labeling heme-binding proteins or glycoproteins. While both heme binding34 and glycoproteins35 have been successfully probed using HRPF, thorough troubleshooting in the concentration of H2O2, radical scavenger, and the timeframe of H2O2 exposure must be balanced. Additionally, some buffers extensively quench ▪OHs. Therefore, it is vital to select an appropriate buffer and limit excipients like DTT and DMSO. Typical buffers used during an HRPF experiment include acetate, sodium phosphate, phosphate buffered saline, or low concentrations of Tris buffer. Interestingly, Tris scavenges some ▪OH, but following oxidation, it increases in photometric absorbance at 265 nm and can be used in place of adenine for dosimetry36. Although some buffers might decrease the effective concentration of ▪OH, by incorporating an in-line radical dosimeter these challenges can be surmounted.
HRPF utilizes non-specific irreversible labeling by ▪OH to probe solvent accessibility. The non-specific nature of the label provides increased overall coverage compared to other more targeted footprinting methods including N-ethylmaleimide37 and glycine ethyl ester38. Since the modification is irreversible, ample time is available during sample handling. This allows for complete protein digestion and a long LC gradient for improved MS/MS analysis. For HRPF there are several methods to generate ▪OH. A popular method utilizes a laser to photolyze H2O2 into ▪OH. Laser based platforms have been very successful in probing native protein structure, but they require extensive safety precautions, rigorous maintenance, and a substantial amount of space.
With the use of a high-pressure plasma source described here, there is no need for harmful gases and no fear of stray UV radiation. The lifetime of each high-pressure plasma source is enough to label between 180-200 samples, and besides monitoring the dosimeter response, no additional maintenance is required. The plasma source is under high-pressure, so while handling the plasma source, wear safety glasses. The plasma source also photolyzes H2O2 more efficiently, allowing for increased protein modification. If insufficient labeling is observed, H2O2 concentration can be increased along with increasing the lamp energy for addition radical production. Additionally, with the incorporation of real-time dosimetry, one can increase the reproducibility of experiments. Altogether, with the use of a plasma source coupled with real-time dosimetry and software for automated FPOP data calculation provides an advantageous package to perform HRPF experiments in a safe, easier to operate, and with improved reproducibility, as compared to standard laser-based methods.
The authors have nothing to disclose.
This work was funded by the National Institute of General Medical Sciences (R43GM125420 and R44GM125420).
15 mL Conical Centrifuge Tubes | Fisher Scientific | 14-959-53A | any brand is sufficient |
50 µL SGE Gastight Syringes | Fisher Scientific | SG-00723 | |
Acclaim PepMap 100 C18 nanocolumn (0.75 mm X 150 mm, 2 µm) | Thermo Scientific | ||
Acetonitrile with 0.1% Formic Acid (v/v), LC/MS Grade | Fisher Scientific | LS120-500 | |
Apomyoglobin | Sigma-Aldrich | ||
Catalase | Sigma-Aldrich | C9322 | |
Centrifuge | Eppendorf | 022625501 | |
Delicate Task Wipers | Fisher Scientific | 06-666A | |
Hydrogen Peroxide | Fisher Scientific | H325-100 | any 30% hydrogen peroxide is sufficient |
Methionine amide | Chem-Impex | 03109 | |
Microcentrifuge | Thermo Scientific | 75002436 | |
Orbitrap Fusion Lumos Tribrid Mass Spectrometer | Thermo Scientific | Orbitrap Fusion Lumos Tribrid Mass Spectrometer | other high resolution instruments (e.g. Q exactive Orbitrap or Orbitrap Fusion) can be used |
Pierce Trypsin Protease, MS Grade | Thermo Scientific | 90058 | |
Polymicro Cleaving Stone, 1" x 1" x 1/32” | Molex | 1068680064 | any capillary tubing cutter is sufficient |
UPLC | Thermo Scientific | ||
Water with 0.1% Formic Acid (v/v), LC/MS Grade | Fisher Scientific | LS118-500 | |
Water, LC/MS Grade | Fisher Scientific | W6-4 |