Development of laser-free, protein oxidation system, real-time radical dosimeter and data processing software for HOS of proteins using HRPF
Year: 2020
Type: article
Abstract: Hydroxyl Radical Protein Foot printing (HRPF) is an emerging higher order structural (HOS) analysis technique. However, its broad-based adoption in the biopharma industry has been limited by critical factors: the use of expensive & dangerous lasers for photolysis; variation among back-ground scavenging HRPF samples; & the extensive and laborious data processing process required to perform the oxidation calculation of peptides. To address these issues, we created novel instrumentation & software for automated calculation of the peptide oxidation. We developed a flash-oxidation (FOXTM) photolysis system that replaces UV lasers with a safe benchtop means to perform laser free HRPF HOS analysis. We validated this instrument by oxidizing apo-myoglobin at various flash energies. Experiments were performed with 1 mM adenine and 100 mM peroxide. An effective real-time optical radical dosimeter, upon reaction with hydroxyl radicals, adenine loses UV absorbance at 265 nm. By monitoring ∆Abs265nm, the effective hydroxyl radical concentration delivered to sample can be determined. To overcome the problem of variation between replicates of the HRPF samples, we developed FOXTM Radical Dosimeter. Real-time measurement of effective hydroxyl radical yield by measuring adenine absorbance provides reproducible labeling irrespective of background scavenging. Singly, doubly, & triply oxidized products of apo-myoglobin were generated. Oxidation products are similar to those generated by laser exposure of the samples. A radical dose dependence plot of peptide oxidation vs radical dosimetry indicated excellent labeling coverage along with the absence of HOS labeling artifacts. A major aspect limiting the wide usage of HRPF is the manual, time-consuming calculation of peptide oxidation & lack of any automated data processing software. We developed FoxWareTM analysis Software for the rapid and reliable quantitation of peptide quantification of HRPF data, encouraging usage in biopharma.
Source: PubMed Central
Authors: Misra, Sandeep, Weinberger, Scot, Orlando, Ron, Sharp, Joshua
Institution: University of Mississippi
Language: English
Subfield: Spectroscopy
Field: Chemistry
Domain: Physical Sciences
Open Access status: green
OpenAlex ID: W3080717366