Skip to main content
SpringerLink
Log in

Conformational Analysis of Therapeutic Proteins by Hydroxyl Radical Protein Footprinting

  • Research Article
  • Theme: Liquid Chromatography Mass Spectrometry for Therapeutic Macromolecules
  • Published:
The AAPS Journal Aims and scope Submit manuscript

Abstract

Unlike small molecule drugs, therapeutic protein pharmaceuticals must not only have the correct amino acid sequence and modifications, but also the correct conformation to ensure safety and efficacy. Here, we describe a method for comparison of therapeutic protein conformations by hydroxyl radical protein footprinting using liquid chromatography-mass spectrometry (LC-MS) as an analytical platform. Hydroxyl radical protein footprinting allows for rapid analysis of the conformation of therapeutic proteins based on the apparent rate of oxidation of various amino acids by hydroxyl radicals generated in situ. Conformations of Neupogen®, a patented granulocyte colony-stimulating factor (GCSF), were compared to several expired samples of recombinant GCSF, as well as heat-treated Neupogen®. Conformations of different samples of the therapeutic proteins interferon α-2A and erythropoietin were also compared. Differences in the hydroxyl radical footprint were measured between Neupogen® and the expired or mishandled GCSF samples, and confirmed by circular dichroism spectroscopy. Samples that had identical circular dichroism spectra were also found to be indistinguishable by hydroxyl radical footprinting. The method is applicable to a wide variety of therapeutic proteins and formulations through the use of separations techniques to clean up the protein samples after radical oxidation. The reaction products are stable, allowing for flexibility in sample handling, as well as archiving and reanalysis of samples. Initial screening can be performed on small amounts of therapeutic protein with minimal training in LC-MS, but samples with structural differences from the reference can be more carefully analyzed by LC-MS/MS to attain higher spatial resolution, which can aid in engineering and troubleshooting.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  1. Locatelli F, Del Vecchio L, Pozzoni P. Pure Red-cell aplasia “epidemic”—mystery completely revealed? Perit Dial Int. 2007;27(Supplement 2):S303–7.

    PubMed  Google Scholar 

  2. McKoy JM, Stonecash RE, Cournoyer D, Rossert J, Nissenson AR, Raisch DW, et al. Epoetin‐associated pure red cell aplasia: past, present, and future considerations. Transfusion. 2008;48(8):1754–62.

    Article  PubMed  Google Scholar 

  3. Kessler M, Goldsmith D, Schellekens H. Immunogenicity of biopharmaceuticals. Nephrol Dial Transplant. 2006;21 suppl 5:v9.

    Article  PubMed  CAS  Google Scholar 

  4. Srebalus Barnes CA, Lim A. Applications of mass spectrometry for the structural characterization of recombinant protein pharmaceuticals. Mass Spectrom Rev. 2007;26(3):370–88.

    Article  PubMed  CAS  Google Scholar 

  5. Bobst CE, Abzalimov RR, Houde D, Kloczewiak M, Mhatre R, Berkowitz SA, et al. Detection and characterization of altered conformations of protein pharmaceuticals using complementary mass spectrometry-based approaches. Anal Chem. 2008;80(19):7473–81.

    Article  PubMed  CAS  Google Scholar 

  6. Kaltashov IA, Bobst CE, Abzalimov RR, Wang G, Baykal B, Wang S. Advances and challenges in analytical characterization of biotechnology products: Mass spectrometry-based approaches to study properties and behavior of protein therapeutics. Biotechnol Adv. 2012;30(1):210–22.

    Article  PubMed  CAS  Google Scholar 

  7. Konermann L, Pan J, Liu YH. Hydrogen exchange mass spectrometry for studying protein structure and dynamics. Chem Soc Rev. 2011;40:1224–34.

    Article  PubMed  CAS  Google Scholar 

  8. Kaltashov IA, Bobst CE, Abzalimov RR, Wang G, Baykal B, Wang S. Advances and challenges in analytical characterization of biotechnology products: Mass spectrometry-based approaches to study properties and behavior of protein therapeutics. Biotechnol Adv. 2012;30(1):210–22.

    Article  PubMed  CAS  Google Scholar 

  9. Mendoza VL, Vachet RW. Probing protein structure by amino acid‐specific covalent labeling and mass spectrometry. Mass Spectrom Rev. 2009;28(5):785–815.

    Article  PubMed  CAS  Google Scholar 

  10. Konermann L, Stocks BB, Pan Y, Tong X. Mass spectrometry combined with oxidative labeling for exploring protein structure and folding. Mass Spectrom Rev. 2010;29(4):651–67.

    PubMed  CAS  Google Scholar 

  11. Xu G, Chance MR. Radiolytic modification and reactivity of amino acid residues serving as structural probes for protein footprinting. Anal Chem. 2005;77(14):4549–55.

    Article  PubMed  CAS  Google Scholar 

  12. Buxton GV, Greenstock CL, Helman WP, Ross AB. Critical review of rate constants for reactions of hydrated electrons, hydrogen atoms and hydroxyl radicals (.Oh/.O−) in aqueous solution. J Phys Chem Ref Data. 1988;17(2):513–886.

    Article  CAS  Google Scholar 

  13. Charvátová O, Foley B, Bern M, Sharp J, Orlando R, Woods R. Quantifying protein interface footprinting by hydroxyl radical oxidation and molecular dynamics simulation: application to galectin-1. J Am Soc Mass Spectrom. 2008;19(11):1692–705.

    Article  PubMed  Google Scholar 

  14. Gau BC, Sharp JS, Rempel DL, Gross ML. Fast photochemical oxidation of protein footprints faster than protein unfolding. Anal Chem. 2009;81(16):6563–71.

    Article  PubMed  CAS  Google Scholar 

  15. Watson C, Janik I, Zhuang T, Charva Tova O, Woods R, Sharp J. Pulsed electron beam water radiolysis for submicrosecond hydroxyl radical protein footprinting. Anal Chem. 2009;81(7):2496–505.

    Article  PubMed  CAS  Google Scholar 

  16. Hambly DM, Gross ML. Laser flash photolysis of hydrogen peroxide to oxidize protein solvent-accessible residues on the microsecond timescale. J Am Soc Mass Spectrom. 2005;16(12):2057–63.

    Article  PubMed  CAS  Google Scholar 

  17. Desjardins P, Hansen JB, Allen M (2009) Microvolume protein concentration determination using the NanoDrop 2000c spectrophotometer. J Visualized Experiments: JoVE (33): 1610

  18. Woody RW. Circular dichroism. Biochem Spectrosc. 1995;246:34–71.

    Article  CAS  Google Scholar 

  19. Zink T, Ross A, Lueers K, Cieslar C, Rudolph R, Holak TA. Structure and dynamics of the human granulocyte colony-stimulating factor determined by NMR spectroscopy. Loop mobility in a four-helix-bundle protein. Biochemistry. 1994;33(28):8453–63.

    Article  PubMed  CAS  Google Scholar 

  20. Ross CA, Poirier MA. Protein aggregation and neurodegenerative disease. Nat Med. 2004;10(Suppl):S10–7.

    Article  PubMed  Google Scholar 

  21. Klaus W, Gsell B, Labhardt AM, Wipf B, Senn H. The three-dimensional high resolution structure of human interferon alpha-2a determined by heteronuclear NMR spectroscopy in solution. J Mol Biol. 1997;274(4):661–75.

    Article  PubMed  CAS  Google Scholar 

  22. Cheetham JC, Smith DM, Aoki KH, Stevenson JL, Hoeffel TJ, Syed RS, et al. NMR structure of human erythropoietin and a comparison with its receptor bound conformation. Nat Struct Biol. 1998;5(10):861–6.

    Article  PubMed  CAS  Google Scholar 

  23. Wang X, Watson C, Sharp JS, Handel TM, Prestegard JH. Oligomeric structure of the chemokine CCL5/RANTES from NMR, MS, and SAXS data. Structure. 2011;19(8):1138–48.

    Article  PubMed  CAS  Google Scholar 

Download references

ACKNOWLEDGMENTS

This project was supported by grants from the National Center for Research Resources (5P41RR005351-23) and the National Institute of General Medical Sciences (8 P41 GM103390-23) from the National Institutes of Health. The content of this work is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. We would also like to thank Dr. Jeffrey Urbauer of The University of Georgia for use of his CD instrumentation. JSS would also like to thank Dr. Marshall Bern for numerous productive conversations on the topics explored here.

Author information

Authors and Affiliations

  1. Complex Carbohydrate Research Center, University of Georgia, 315 Riverbend Rd, Athens, Georgia, 30602, USA

    Caroline Watson & Joshua S. Sharp

Authors
  1. Caroline Watson
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Joshua S. Sharp
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Joshua S. Sharp.

Additional information

Guest Editor: Michael Bartlett

Electronic supplementary material

Below is the link to the electronic supplementary material.

Table S1

Independent two-tailed Student’s t test of HRF between GCSF samples (DOC 34 kb)

Table S2

Independent two-tailed Student’s t test of HRF between IFN samples and IFN 2005 (DOC 28 kb)

Table S3

Independent two-tailed Student’s t test of HRF between EPO samples and EPO June 2009 (DOC 26 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Watson, C., Sharp, J.S. Conformational Analysis of Therapeutic Proteins by Hydroxyl Radical Protein Footprinting. AAPS J 14, 206–217 (2012). https://doi.org/10.1208/s12248-012-9336-7

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1208/s12248-012-9336-7

Key words

Search

Navigation