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 language | English |
|---|---|
| Pages (from-to) | 987-992 |
| Number of pages | 6 |
| Journal | Biochemistry |
| Volume | 45 |
| Issue number | 3 |
| DOIs | |
| Publication status | Published - Jan 24 2006 |
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- Biochemistry
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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 journal › Article
}
TY - JOUR
T1 - Stepwise deamidation of ribonuclease A at five sites determined by top down mass spectrometry
AU - Zabrouskov, Vlad
AU - Han, Xuemei
AU - Welker, E.
AU - Zhai, Huili
AU - Lin, Cheng
AU - Van Wijk, Klaas J.
AU - Scheraga, Harold A.
AU - McLafferty, Fred W.
PY - 2006/1/24
Y1 - 2006/1/24
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=31044442964&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=31044442964&partnerID=8YFLogxK
U2 - 10.1021/bi0517584
DO - 10.1021/bi0517584
M3 - Article
C2 - 16411774
AN - SCOPUS:31044442964
VL - 45
SP - 987
EP - 992
JO - Biochemistry
JF - Biochemistry
SN - 0006-2960
IS - 3
ER -