Format

Send to

Choose Destination
J Am Soc Mass Spectrom. 2018 Sep;29(9):1901-1907. doi: 10.1007/s13361-018-1994-y. Epub 2018 Jun 25.

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

Author information

1
Department of BioMolecular Sciences, School of Pharmacy, University of Mississippi, University, MS, 38655, USA.
2
Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, New York, NY, 10016, USA.
3
Howard Hughes Medical Institute, New York University School of Medicine, New York, NY, 10016, USA.
4
Department of BioMolecular Sciences, School of Pharmacy, University of Mississippi, University, MS, 38655, USA. jsharp@olemiss.edu.

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

PMID:
29943081
PMCID:
PMC6087495
[Available on 2019-09-01]
DOI:
10.1007/s13361-018-1994-y

Supplemental Content

Full text links

Icon for Springer
Loading ...
Support Center