Journal of The American Society for Mass Spectrometry

, Volume 29, Issue 9, pp 1901–1907 | Cite as

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

  • Niloofar Abolhasani Khaje
  • Charles K. Mobley
  • Sandeep K. Misra
  • Lindsey Miller
  • Zixuan Li
  • Evgeny Nudler
  • Joshua S. SharpEmail author
Research Article


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


Hydroxyl radical protein footprinting FPOP Protein oxidation 



The authors thank Dr. Kelley Moremen for the expression and purification of COSMC and RPTP Sigma, Dr. Thomas Clausen for the expression and purification of VAR2CSA, and Dr. Christopher West for the expression and purification of Skp1. E.N. acknowledges support of the Howard Hughes Medical Institute and the Blavatnik Family Foundation.

Funding Information

This research was supported by the National Institute of General Medical Sciences Research Resource for Integrated Glycotechnology (P41GM103390) and the National Science Foundation (CHE1608685).

Supplementary material

13361_2018_1994_MOESM1_ESM.docx (127 kb)
ESM 1 (DOCX 126 kb)


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Copyright information

© American Society for Mass Spectrometry 2018

Authors and Affiliations

  • Niloofar Abolhasani Khaje
    • 1
  • Charles K. Mobley
    • 1
  • Sandeep K. Misra
    • 1
  • Lindsey Miller
    • 1
  • Zixuan Li
    • 2
    • 3
  • Evgeny Nudler
    • 2
    • 3
  • Joshua S. Sharp
    • 1
    Email author
  1. 1.Department of BioMolecular Sciences, School of PharmacyUniversity of MississippiUniversityUSA
  2. 2.Department of Biochemistry and Molecular PharmacologyNew York University School of MedicineNew YorkUSA
  3. 3.Howard Hughes Medical InstituteNew York University School of MedicineNew YorkUSA

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