Detection and Measurement of Methionine Oxidation in Proteins

K. Ilker Sen1, Robert Hepler2, Hirsh Nanda2

1 Protein Metrics Inc, San Carlos, 2 Janssen Research & Development, Pennsylvania
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 14.16
DOI:  10.1002/cpps.25
Online Posting Date:  February, 2017
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Abstract

Methionine oxidation is a prevalent modification found in proteins both in biological settings and in the manufacturing of biotherapeutic molecules. In cells, the oxidation of specific methionine sites can modulate protein function or promote interactions that trigger signaling pathways. In biotherapeutic development, the formation of oxidative species could be detrimental to the efficacy or safety of the drug product. Thus, methionine oxidation is a critical quality attribute that needs to be monitored throughout development. Here we describe a method using LC/MS/MS to identify site‐specific methionine modifications in proteins. Antibodies are stressed with hydrogen peroxide, and the level of Met oxidation is compared to that of reference molecules. The protocols presented here are not specific to methionine and can be used more generally to identify other PTM risk sites in molecules after various types of treatments. © 2017 by John Wiley & Sons, Inc.

Keywords: oxidation; mass spectrometry; protein; Biopharmaceutical; Byonic; Byologic

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Identification and Quantification of Methionine Oxidation in Proteins
  • Alternate Protocol 1: Forced Stress of Proteins by Chemical Oxidation
  • Alternate Protocol 2: Forced Stress of Proteins by Thermal Stress
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Identification and Quantification of Methionine Oxidation in Proteins

  Materials
  • Sample: protein is typically supplied purified, in PBS, pH 7.4, at 1 mg/ml concentration (other buffers are acceptable since the protocol 1Basic Protocol involves a buffer‐exchange step; lower concentration sample is also acceptable, depending on the sensitivity of the LC/MS apparatus used)
  • 8 M guanidine hydrochloride solution, buffered, pH 8.5 (Sigma‐Aldrich, cat. no. G7294)
  • 0.5 M EDTA solution, pH 8.0 (Sigma‐Aldrich, cat. no. 03690)
  • 1 M dithiothreitol (DTT; Sigma‐Aldrich, cat. no. 646563)
  • Iodoacetamide (Sigma‐Aldrich, cat. no. A3221)
  • Digestion buffer: 50 mM Tris·Cl, pH 8.0/1 mM CaCl 2 (prepare from 1 M Tris and CaCl 2 stocks)
  • 1 mg/ml trypsin (sequencing grade, Promega) in 50 mM acetic acid
  • 100% trifluoroacetic acid (TFA; Sigma‐Aldrich)
  • 0.1% (v/v) formic acid in H 2O, LC/MS grade (Honeywell)
  • Acetonitrile, LC/MS grade (Honeywell)
  • 0.5‐ml Spin Desalting Column (Zeba, Thermo Fisher)
  • Agilent 1290 or Waters Acquity UPLC coupled with an Agilent AdvanceBio Peptide Mapping Column (1.0 × 150 mm, 2.7 µm particle)
  • Electrospray ionization mass spectrometer (e.g., Thermo Orbitrap Fusion Lumos or Q‐Exactive Plus, Waters Synapt G2‐Si or Xevo G2‐XS, SCIEX TripleTOF 6600, Agilent 6500 series Q‐TOF, Bruker maXis II or 7T solariX, or similar instrument; also see Chapter 16)
  • MS/MS search engine (Byonic, Protein Metrics, Inc.)
  • Additional reagents and equipment for mass spectrometry (Chapter 16)

Alternate Protocol 1: Forced Stress of Proteins by Chemical Oxidation

  Materials
  • Protein sample
  • 30% (w/w) hydrogen peroxide solution in H 2O (Sigma‐Aldrich)
  • Catalase from bovine liver (Sigma‐Aldrich)

Alternate Protocol 2: Forced Stress of Proteins by Thermal Stress

  Materials
  • Protein sample
  • Phosphate‐buffered saline (PBS), pH 7.4 (VWR)
  • Amicon Ultra‐15 Centrifugal Filter Units with Ultracel‐30 membrane (EMD Millipore)
  • SoloVPE variable path‐length spectrometer (C Technologies, Inc.)
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Figures

Videos

Literature Cited

  
  Guan, Z., Yates, N.A., and Bakhtiar, R. 2003. Detection and characterization of methionine oxidation in peptides by collision‐induced dissociation and electron capture dissociation. J. Am. Soc. Mass Spectrom. 14:605‐613. doi: 10.1016/S1044‐0305(03)00201‐0.
  Hao, P. and Sze, S.K. 2014. Proteomic analysis of protein deamidation. Curr. Protoc. Protein Sci. 78:24.5.1‐24.5.14. doi: 10.1002/0471140864.ps2405s78.
  Hollemeyer, K., Heinzle, E., and Tholey, A. 2002. Identification of oxidized methionine residues in peptides containing two methionine residues by derivatization and matrix‐assisted laser desorption/ionization mass spectrometry. Proteomics 2:1524‐1531. doi: 10.1002/1615‐9861(200211)2:11%3c1524::AID‐PROT1524%3e3.0.CO;2‐7.
  Hoshi, T. and Heinemann, S. 2001. Regulation of cell function by methionine oxidation and reduction. J. Physiol. 531:1‐11. doi: 10.1111/j.1469‐7793.2001.0001j.x.
  Levine, R.L., Moskovitz, J., and Stadtman, E.R. 2000. Oxidation of methionine in proteins: Roles in antioxidant defense and cellular regulation. IUBMB Life 50:301‐307. doi: 10.1080/15216540051081056.
  Li, W., Kerwin, J.L., Schiel, J., Formolo, T., Davis, D., Mahan, A., and Benchaar, S.A. 2015. Structural elucidation of post‐translational modifications in monoclonal antibodies. In State‐of‐the‐Art and Emerging Technologies for Therapeutic Monoclonal Antibody Characterization, Volume 2, Biopharmaceutical Characterization: The NISTmAb Case Study, vol. 1201 (J.E. Schiel, D.L. Davis, and O.V. Borisov, eds.), pp. 119‐183. American Chemical Society, Washington, D.C.
  Maier, K. L., Lenz, A. G., Beck‐Speier, I., and Costabel, U. 1995. Analysis of methionine sulfoxide in proteins. Methods Enzymol. 251:455‐461. doi: 10.1016/0076‐6879(95)51149‐0.
  Saro, D., Baker, A., Hepler, R., Spencer, S., Bruce, R., LaBrenz, S., Chiu, M., Davis, D., and Lang, S.E. 2015. Developability assessment of a proposed NIST monoclonal antibody. In State‐of‐the‐Art and Emerging Technologies for Therapeutic Monoclonal Antibody Characterization, Volume 2, Biopharmaceutical Characterization: The NISTmAb Case Study, Chapter 3 (J.E. Schiel, D.L. Davis, and O.V. Borisov, eds.), pp. 119‐183. American Chemical Society, Washington, D.C.
  Schey, K.L. and Finley, E.L. 2000. Identification of peptide oxidation by tandem mass spectrometry. Acc. Chem. Res. 33:299‐306. doi: 10.1021/ar9800744.
  Stadtman, E.R. and Berlett, B.S. 1998. Reactive oxygen‐mediated protein oxidation in aging and disease. Drug Metab. Rev. 30:225‐243. doi: 10.3109/03602539808996310.
  Wang, W., Vlasak, J., Li, Y., Pristatsky, P., Fang, Y., Pittman, T., Roman, J., Wang, Y., Prueksaritanont, T., and Ionescu, R. 2011. Impact of methionine oxidation in human IgG1 Fc on serum half‐life of monoclonal antibodies. Mol. Immunol. 48:860‐866. doi: 10.1016/j.molimm.2010.12.009.
Key References
  Josic, D. and Kovac, S. 2010. Reversed‐phase high performance liquid chromatography of proteins. Curr. Protoc. Protein Sci. 61:8.7.1‐8.7.22. doi: 10.1002/0471140864.ps0807s61.
  RP‐HPLC is in indispensable tool in protein and peptide LC/MS applications, as it is readily compatible with mass spectrometry. This Current Protocols in Protein Science unit details protocols for RP‐HPLC that are used in the present unit.
  Zhang, G., Annan, R.S., Carr, S.A. and Neubert, T.A. 2010. Overview of peptide and protein analysis by mass spectrometry. Curr. Protoc. Protein Sci. 62:16.1.1‐16.1.30. doi: 10.1002/0471140864.ps1601s62.
  Introductory overview of mass spectrometry of proteins, including considerations for instruments and experimental workflows.
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