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Identification of Asp Isomerization in Proteins by 18O Labeling and Tandem Mass Spectrometry

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Therapeutic Proteins

Part of the book series: Methods in Molecular Biology ((MIMB,volume 899))

Abstract

Isomerization of aspartic acid (Asp) to isoaspartic acid (isoAsp) via succinimide intermediate is a common route of degradation for proteins that can affect their structural integrity. As Asp/isoAsp is isobaric in mass, it is difficult to identify the site of modification by LC-MS/MS peptide mapping. Here, we describe an approach to label the Asp residue involved in isomerization at the protein level by hydrolyzing the succinimide intermediate in H 182 O. Tryptic digestion of this labeled protein will result in peptides containing the site of isomerization being 2 Da heavier than the 16O-containing counterparts, due to 18O incorporation during the hydrolysis process. Comparison of tandem mass spectra of isomerized peptides with and without 18O incorporation allows easy identification of the Asp residue involved. This method proved to be especially useful in identifying the sites when isomerization occurs in Asp-Asp motifs.

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Acknowledgments

The authors gratefully acknowledge Clifford Quan for synthesis of peptide variants and Benson Gikanga for preparing the thermally stressed samples. We would also like to thank Reed Harris, John Stults, Mary Cromwell, and Charles Morgan for their valuable discussion and critical review.

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Authors and Affiliations

  1. Protein Analytical Chemistry, Genentech Inc, South San Francisco, CA, USA

    Jennifer Zhang & Viswanatham Katta

Authors
  1. Jennifer Zhang
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  2. Viswanatham Katta
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Correspondence to Jennifer Zhang .

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Editors and Affiliations

  1. MedImmune, LLC, MedImmune Way One, Gaithersburg, 20878, USA

    Vladimir Voynov

  2. , Department of Physical Biochemistry, Biogen Idec, Cambridge Center 14, Cambridge, 02142, Massachusetts, USA

    Justin A. Caravella

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Zhang, J., Katta, V. (2012). Identification of Asp Isomerization in Proteins by 18O Labeling and Tandem Mass Spectrometry. In: Voynov, V., Caravella, J. (eds) Therapeutic Proteins. Methods in Molecular Biology, vol 899. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-921-1_23

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  • DOI: https://doi.org/10.1007/978-1-61779-921-1_23

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-61779-920-4

  • Online ISBN: 978-1-61779-921-1

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