Stepwise deamidation of ribonuclease A at five sites determined by top down mass spectrometry.
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.
Zabrouskov
Vlad
V
Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York 14853, USA.
Han
Xuemei
X
Welker
Ervin
E
Zhai
Huili
H
Lin
Cheng
C
van Wijk
Klaas J
KJ
Scheraga
Harold A
HA
McLafferty
Fred W
FW
D000577:Amides / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D008969:Molecular Sequence Data; D010179:Pancreas / Q000201:enzymology*; D012259:Ribonuclease, Pancreatic / Q000737:chemistry* / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization
D000577:Amides; D012259:Ribonuclease, Pancreatic