Identification of modification sites in large biomolecules by stable isotope labeling and tandem high resolution mass spectrometry. The active site nucleophile of thiaminase I.
A widely used procedure for site localization of covalent protein modifications involves proteolysis, partial chromatographic separation of the resulting complex mixture, and tandem mass spectrometry (MS/MS) to identify peptides whose molecular weight (Mr) has been increased appropriately by the modification. As found previously for MS of small molecules, this study shows that protein fragment identification can be greatly simplified by labeling the modification with stable isotopes. Further, the high resolution capabilities of Fourier transform MS make possible the direct identification of CH3/CD3-labeled peptides without chromatographic separation. Although separate Asp-N, Lys-C, and alpha-chymotrypsin digests of thiaminase I (42 kDa) yielded as many as 70 peptides, FTMS identification of the labeled peptide localized the modification site of a mechanism-based inhibitor to Arg101-Lys121, Asp90-Gly122, and Gly107-Tyr119, respectively. The measured mass difference values of the two labels agreed with that expected for CH3/CD3, 3.019 Da, with a standard deviation of 0.005 Da, providing persuasive identity verification. MS/MS fragmentation narrowed the site to Pro109-Phe118 and also caused loss of the derivative with a sulfur atom, uniquely identifying Cys113 as the thiaminase I active-site nucleophile among the 379 amino acids.
Kelleher
N L
NL
Department of Chemistry, Baker Laboratory, Cornell University, Ithaca, New York 14853-1301, USA.
Nicewonger
R B
RB
Begley
T P
TP
McLafferty
F W
FW
D019883:Alkyl and Aryl Transferases / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D003545:Cysteine / Q000737:chemistry; D007553:Isotope Labeling; D013058:Mass Spectrometry / Q000379:methods
D019883:Alkyl and Aryl Transferases; C022737:thiamin pyridinylase; D003545:Cysteine