Protein footprinting is an approach to determine changes in proteins resulting from binding, aggregation, and various other perturbations. These perturbations modify the solvent accessibility of certain regions (e.g., binding interfaces) of the protein. These changes can be probed by chemical reactions that occur more slowly when the region becomes less solvent-accessible (1).
Fast photochemical oxidation of proteins (FPOP) is a MS-based protein footprinting method that was developed at this research resource (2, 3). FPOP utilizes a pulsed laser to photolyze hydrogen peroxide to generate OH radicals and modify proteins in a flow system on the microsecond timescale (3). The laser provides a spatially small, high flux of light, maximizing the exposure of a small volume of protein solution to radicals and ensuring all but a small fraction of the protein in the flow is irradiated only once.
About fourteen out of twenty amino acids can be labeled upon laser exposure during FPOP. Their side chains are modified by reacting with •OH, with different amino acids exhibiting different rate constants and modification mechanisms. A typical side chain modification is the substitution of an OH for H, which results in a +16 Da to the total mass of the protein.
This method is advantageous in that: (i) this stable covalent modification is irreversible, which preserves the primary sequence of modified residues regardless of subsequent sample handling and proteolysis; (ii) the high reactivity of •OH towards amino acids enables the modifications of more than half of amino acid side-chains, providing a higher coverage than other approaches that only target specific residues (e.g., the acetylation of primary amines); (iii) the size of •OH is comparable to a water molecule, rendering this approach able to probe the solvent accessibility of a protein of interest.
FPOP is performed as shown in the figure above. Samples with an exclusion volume fraction of 20% (the volume not irradiated by laser, sandwiched in between plugs of the irradiated solution) are advanced at a constant rate by syringe pump. The excimer laser power is ~45 mJ/pulse. Samples then flow into collection vials containing catalase and methionine that destroy residual amounts of hydrogen peroxide.
Because FPOP is rapid and irreversible, it can be used to study protein folding when coupled with other methods. An example is the two-color experiment that couples a temperature jump with FPOP (4, 5). This is a new MS-based approach for studying protein-folding dynamics at the amino-acid level on the sub-millisecond time scale. Two lasers are incorporated in this approach, one to provide a temperature jump altering the protein’s equilibrium conformation (Nd:YAG laser) and the other to generate hydroxyl radicals (excimer laser) to footprint the consequence of the T jump. The delay between the Nd:YAG and excimer lasers can be made as short as the radical lifetime in FPOP, which is ~1 μs. The change in reaction between side chain residues and OH radicals as a function of delay time serves as a reporter of the conformational change. This method enables one to reveal the sites and kinetics of protein folding/unfolding on the sub-millisecond time scale. Kinetics on microsecond time scale can also be reached, due to the pulse widths of the two lasers (10 ns for the Nd:YAG laser and 20 ns for the excimer laser) and the quenching time for hydroxyl radicals (<1 µs).
The combination of a fast mixing device with FPOP can also be a useful tool to probe the
structural and conformational changes upon protein folding. Typically we use two syringes, one containing protein solution and the other a denaturant or binding ligand. Both syringes are advanced by a syringe pump, the solutions and the solutions are mixed in a tee. With this setup one can measure folding events on the millisecond time scale. Microfluidic mixing devices will make the sub-millisecond time scale reachable. Following fast folding or unfolding of proteins is a research goal of the WU Research Resource.
Another advantage of FPOP is that other reagents besides OH radicals can be used. Examples are the sulfate radical cation (6) and the iodide radical (7). The iodide radical is considerable more specific than ∙OH, reacting with His and Tyr residues only. Development of new reagents is another goal of the Resource.
Standing in the way of widespread use of FPOP is data processing, and Resource scientists are also working on methods to solve this need (8).
- Xu, G., and Chance, M. R. (2007) Hydroxyl radical-mediated modification of proteins as probes for structural proteomics, Chemical Reviews 107, 3514-3543.
- Hambly, D. M., and Gross, M. L. (2005) Laser flash photolysis of hydrogen peroxide to oxidize protein solvent-accessible residues on the microsecond timescale, J Am Soc Mass Spectrom 16, 2057-2063.
- Gau, B. C., Sharp, J. S., Rempel, D. L., and Gross, M. L. (2009) Fast photochemical oxidation of protein footprints faster than protein unfolding, Anal Chem 81, 6563-6571.
- Chen, J., Rempel, D. L., and Gross, M. L. (2010) Temperature jump and fast photochemical oxidation probe submillisecond protein folding, J Am Chem Soc 132, 15502-15504.
- Chen, J., Rempel, D. L., Gau, B. C., and Gross, M. L. (2012) Fast photochemical oxidation of proteins and mass spectrometry follow submillisecond protein folding at the amino-acid level, Journal of the American Chemical Society 134, 18724-18731.
- Gau, B. C., Chen, H., Zhang, Y., and Gross, M. L. Sulfate radical anion as a new reagent for fast photochemical oxidation of proteins, Analytical Chemistry 82, 7821-7827.
- Chen, J., Cui, W., Giblin, D., and Gross, M. L. (2012) New protein footprinting: fast photochemical iodination combined with top-down and bottom-up mass spectrometry, J Am Soc Mass Spectrom 23, 1306-1318.
- Gau, B. C., Chen, J., and Gross, M. L. (2013) Fast photochemical oxidation of proteins for comparing solvent- accessibility changes accompanying protein folding: Data processing and application to barstar, Biochimica et Biophysica Acta – Proteins and Proteomics 1834, 1230-1238.