### Probing the Time Scale of FPOP (Fast Photochemical Oxidation of Proteins): Radical Reactions Extend Over Tens of MillisecondsProbing the Time Scale of FPOP (Fast Photochemical Oxidation of Proteins): Radical Reactions Extend Over Tens of Milliseconds

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 Author Vahidi, Siavash ♦ Konermann, Lars Source SpringerLink Content type Text Publisher Springer US File Format PDF Copyright Year ©2016 Language English
 Subject Domain (in DDC) Natural sciences & mathematics ♦ Chemistry & allied sciences Subject Keyword Protein structure ♦ Oxidative labeling ♦ Flash photolysis ♦ Dye bleaching ♦ Radical chemistry ♦ Analytical Chemistry ♦ Biotechnology ♦ Organic Chemistry ♦ Proteomics ♦ Bioinformatics Abstract Hydroxyl radical (⋅OH) labeling with mass spectrometry detection reports on protein conformations and interactions. Fast photochemical oxidation of proteins (FPOP) involves ⋅OH production via H$_{2}$O$_{2}$ photolysis by UV laser pulses inside a flow tube. The experiments are conducted in the presence of a scavenger (usually glutamine) that shortens the ⋅OH lifetime. The literature claims that FPOP takes place within 1 μs. This ultrafast time scale implies that FPOP should be immune to labeling-induced artifacts that may be encountered with other techniques. Surprisingly, the FPOP time scale has never been validated in direct kinetic measurements. Here we employ flash photolysis for probing oxidation processes under typical FPOP conditions. Bleaching of the reporter dye cyanine-5 (Cy5) served as readout of the time-dependent radical milieu. Surprisingly, Cy5 oxidation extends over tens of milliseconds. This time range is four orders of magnitude longer than expected from the FPOP literature. We demonstrate that the glutamine scavenger generates metastable secondary radicals in the FPOP solution, and that these radicals lengthen the time frame of Cy5 oxidation. Cy5 and similar dyes are widely used for monitoring the radical dose experienced by proteins in solution. The measured Cy5 kinetics thus strongly suggest that protein oxidation in FPOP extends over a much longer time window than previously thought (i.e., many milliseconds instead of one microsecond). The optical approach developed here should be suitable for assessing the performance of future FPOP-like techniques with improved temporal labeling characteristics. Graphical Abstract ᅟ ISSN 10440305 Age Range 18 to 22 years ♦ above 22 year Educational Use Research Education Level UG and PG Learning Resource Type Article Publisher Date 2016-04-11 Publisher Institution The American Society for Mass Spectrometry Publisher Place New York e-ISSN 18791123 Journal Journal of The American Society for Mass Spectrometry Volume Number 27 Issue Number 7 Page Count 9 Starting Page 1156 Ending Page 1164