Variation in FPOP Measurements Is Primarily Caused by Poor Peptide Signal Intensity

Abstract

Fast photochemical oxidation of proteins (FPOP) may be used to characterize changes in protein structure by measuring differences in the apparent rate of peptide oxidation by hydroxyl radicals. The variability between replicates is high for some peptides and limits the statistical power of the technique, even using modern methods controlling variability in radical dose and quenching. Currently, the root cause of this variability has not been systematically explored, and it is unknown if the major source(s) of variability are structural heterogeneity in samples, remaining irreproducibility in FPOP oxidation, or errors in LC-MS quantification of oxidation. In this work, we demonstrate that coefficient of variation of FPOP measurements varies widely at low peptide signal intensity, but stabilizes to ≈ 0.13 at higher peptide signal intensity. We dramatically reduced FPOP variability by increasing the total sample loaded onto the LC column, indicating that the major source of variability in FPOP measurements is the difficulties in quantifying oxidation at low peptide signal intensities. This simple method greatly increases the sensitivity of FPOP structural comparisons, an important step in applying the technique to study subtle conformational changes and protein-ligand interactions. Graphical Abstract ᅟ.

Keywords: FPOP; Hydroxyl radical protein footprinting; Protein oxidation.

Figure 1

OEP CV versus the ΣPI of 180 oxidized peptides from ten model proteins. As the signal intensity of a peptide gets smaller, the variability in the amount of oxidation measured from triplicate samples increases.

Figure 2

Effect of injection amount on the oxidation event per peptide measurements of three different proteins: (Top) hen egg white lysozyme, (Middle) horse heart myoglobin, and (Bottom) COSMC. Solid and outlined columns represent 2 and 5μL injection volume, respectively. Error bars represent the oxidation events per peptide standard deviation. A two-tailed Student’s t-test was performed to test statistical significance between the OEP of each peptide at two different injection amounts, and found no statistically significant differences (α = 0.05).

Figure 3

OEP CV versus the ΣPI of COSMC peptides before the removal of the suspected misidentified peptides. Peptide 1 and 2 are suspected misidentified peptides.

Figure 4

Mass error of all oxidized and unoxidized of all COSMC peptides. The red columns represent the hypothetically misidentified peptides with an aberrantly large mass error, while the blue ones represent the peptides with a mass error of <7 ppm.