Stepwise deamidation of ribonuclease A at five sites determined by top down mass spectrometry

Vlad Zabrouskov, Xuemei Han, E. Welker, Huili Zhai, Cheng Lin, Klaas J. Van Wijk, Harold A. Scheraga, Fred W. McLafferty

Research output: Contribution to journalArticle

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Abstract

Although deamidation at asparagine and glutamine has been found in numerous studies of a variety of proteins, in almost all cases the analytical methodology that was used could detect only a single site of deamidation. For the extensively studied case of reduced bovine ribonuclease A (13 689 Da), only Asn67 deamidation has been demonstrated previously, although one study found three monodeamidated fractions. Here top down tandem mass spectrometry shows that Asn67 deamidation is extensive before Asn71 and Asn94 react; these are more than half deamidated before Asn34 reacts, and its deamidation is extensive before that at Gln74 is initiated. Except for the initial Asn67 site, these large reactivity differences correlate poorly with neighboring amino acid identities and instead indicate residual conformational effects despite the strongly denaturing media that were used; deamidation at Asn67 could enhance that at Asn71, and these enhance that at Gln74. This success in the site-specific quantitation of deamidation in a 14 kDa protein mixture, despite the minimal 1 Da (-NH2 → -OH) change in the molecular mass, is further evidence of the broad applicability of the top down MS/MS methodology for characterization of protein posttranslational modifications.

Original languageEnglish
Pages (from-to)987-992
Number of pages6
JournalBiochemistry
Volume45
Issue number3
DOIs
Publication statusPublished - Jan 24 2006

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Pancreatic Ribonuclease
Mass spectrometry
Mass Spectrometry
Proteins
Asparagine
Molecular mass
Post Translational Protein Processing
Tandem Mass Spectrometry
Glutamine
Amino Acids

ASJC Scopus subject areas

  • Biochemistry

Cite this

Zabrouskov, V., Han, X., Welker, E., Zhai, H., Lin, C., Van Wijk, K. J., ... McLafferty, F. W. (2006). Stepwise deamidation of ribonuclease A at five sites determined by top down mass spectrometry. Biochemistry, 45(3), 987-992. https://doi.org/10.1021/bi0517584

Stepwise deamidation of ribonuclease A at five sites determined by top down mass spectrometry. / Zabrouskov, Vlad; Han, Xuemei; Welker, E.; Zhai, Huili; Lin, Cheng; Van Wijk, Klaas J.; Scheraga, Harold A.; McLafferty, Fred W.

In: Biochemistry, Vol. 45, No. 3, 24.01.2006, p. 987-992.

Research output: Contribution to journalArticle

Zabrouskov, V, Han, X, Welker, E, Zhai, H, Lin, C, Van Wijk, KJ, Scheraga, HA & McLafferty, FW 2006, 'Stepwise deamidation of ribonuclease A at five sites determined by top down mass spectrometry', Biochemistry, vol. 45, no. 3, pp. 987-992. https://doi.org/10.1021/bi0517584
Zabrouskov, Vlad ; Han, Xuemei ; Welker, E. ; Zhai, Huili ; Lin, Cheng ; Van Wijk, Klaas J. ; Scheraga, Harold A. ; McLafferty, Fred W. / Stepwise deamidation of ribonuclease A at five sites determined by top down mass spectrometry. In: Biochemistry. 2006 ; Vol. 45, No. 3. pp. 987-992.
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