Stepwise Deamidation of Ribonuclease A at Five Sites Determined by Top Down Mass Spectrometry

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• 作者：Vlad Zabrouskov ; Xuemei Han ; Ervin Welker ; Huili Zhai ; Cheng Lin ; Klaas J. van Wijk ; Harold A. Scheraga ; and Fred W. McLafferty
• 刊名：Biochemistry
• 出版年：2006
• 出版时间：January 24, 2006
• 年：2006
• 卷：45
• 期：3
• 页码：987 - 992
• 全文大小：126K
• 年卷期：v.45,no.3(January 24, 2006)
• ISSN：1520-4995

文摘

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