Towards high-throughput fast photochemical oxidation of proteins: Quantifying exposure in high fluence microtiter plate photolysis

Abstract

Protein structural analysis by mass spectrometry has gained significant popularity in recent years, including high-resolution protein topographical mapping by fast photochemical oxidation of proteins (FPOP). The ability to provide protein topographical information at moderate spatial resolution makes FPOP an attractive technology for the protein pharmaceutical discovery and development processes. However, current technology limits the throughput and requires significant manual sample manipulation. Similarly, as FPOP is being used on larger samples, sample flow through the capillary becomes challenging. No systematic comparison of the performance of static flash photolysis with traditional flow FPOP has been reported. Here, we evaluate a 96-well microtiter-based laser flash photolysis method for the topographical probing of proteins, which subsequently could be used to analyze higher order structure of the protein in a high-throughput fashion with minimal manual sample manipulation. We used multiple metrics to compare microtiter FPOP performance with that of traditional flow FPOP: adenine-based hydroxyl radical dosimetry, oxidation efficiency of a model peptide, and hydroxyl radical protein footprint of myoglobin. In all cases, microtiter plate FPOP performed comparably with traditional flow FPOP, requiring a small fraction of the time for exposure. This greatly reduced sample exposure time, coupled with automated sample handling in 96-well microtiter plates, makes microtiter-based FPOP an important step in achieving the throughput required to adapt hydroxyl radical protein footprinting for screening purposes.

Keywords: Covalent labeling; Fast photochemical oxidation of proteins (FPOP); Hydroxyl radical protein footprinting (HRPF); Mass spectrometry; Myoglobin.

Figure 1:. Schematic cartoon for microtiter FPOP.

A mirror placed at 45⁰ angles of laser path deflects the laser beam onto a lens, which then focuses the light onto the well of the microtiter plate.

Figure 2.. Adenine dosimetry of microtiter FPOP.

(A): Representative data set of microtiter single pulse photolysis carried out in a flat-bottomed 96-well microtiter plate. (B) Microtiter single pulse photolysis carried out in a (orange) V-shaped 96-well microtiter plate or (blue) traditional flow FPOP in a fused silica capillary. Error bars represent one standard deviation from a triplicate data set.

Figure 3.. Comparison of GluB peptide oxidation by (orange) microtiter single pulse photolysis versus (blue) traditional capillary FPOP.

Error bars represent one standard deviation from a triplicate data set.

Fig 4.. Correlation among oxidation event for seven peptides of myoglobin.

5 µM myoglobin exposed to four different laser fluences keeping reagent concentration constant. Error bars represent one standard deviation from a triplicate data set. Peptides are grouped according to level of oxidation for improved clarity of data; both left and right panels represent peptides from the same set of experiments.

Fig 5.. Comparison of myoglobin peptide oxidation between (blue) capillary FPOP and (orange) microtiter single pulse photolysis.

Error bars represent one standard deviation from a triplicate data set. No statistically significant differences in oxidation were detected (α = 0.05).