Precise control of OH radical yield with the Fox Protein Footprinting System
GenNext has obsoleted the use of expensive, complicated, and hazardous Class IV Excimer and Nd:YAG lasers with our proprietary high energy plasma photolysis source that creates impressive OH radical loads at a fraction of the cost.
As is the case for conventional Fast Photo-Oxidation of Proteins (FPOP) for Hydroxyl Radical Protein Footprinting (HRPF), our Fox Protein Footprinting System photo-catalyzes the creation of OH radicals from H2O2, but in a highly controlled and cost-effective manner. Unlike laser systems, photolysis fluence can be precisely controlled to achieve desired OH radical yield. The interplay between photolysis fluence and OH radical yield as determined by radical dosimetry is shown in Figure 1. Fluence is regulated at precision better than +/-5 μJ per square millimeter. The photolysis pulse width is precisely controlled to less than 15 μsec, ensuring that foot-printing proceeds without the creation of unwanted HOS artifacts, typically seen in longer-term labeling approaches.
Results obtained with Fox System are eminently consistent with those of conventional FPOP, as demonstrated by the labeling and mass spectrometric analysis of Angiotensin II and protein Apo-Myoglobin. Angiotensin II was labeled with the Fox Photolysis System: 1 μM aqueous solution of Angiotensin II containing 100 mM of H2O2, 1 mM Adenine (radical dosimeter internal standard) in 100 mM phosphate buffer. The sample was irradiated using 6 mJ per square millimeter fluence. The resultant peptide profile is shown in Figure 2. As can be seen, singly oxidized and doubly oxidized products were formed, shifting the average monoisotopic mass of the labeled Angiotensin II to 529.221 m/z, consistent with results obtained by conventional FPOP (> 527 m/z).
Apo-Myoglobin was labeled in a similar fashion as Angiotensin II. As shown in Figure 3, singly, doubly, and triply oxidized products were produced. The Poisson distribution for each species indicates the absence of artifactual changes in protein HOS highly consistent with classical FPOP [15, 17, 22].
Fig. 3: Fox HRPF of Apo-Myoglobin
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