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 modi fications involves proteolysis, partial chromatographic separation of the resulting complex mixture, and tandem mass spectrometry (MS/MS) to identify peptides whose molecular weight (M-r) has been increased app ropriately by the modification, As found previously for MS of small mo lecules, 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 withou t chromatographic separation. Although separate Asp-N, Lys-C, and alph a-chymotrypsin digests of thiaminase I (42 kDa) yielded as many as 70 peptides, FTMS identification of the labeled peptide localized the mod ification site of a mechanism-based inhibitor to Arg(101)-Lys(121), As p(90)-Gly(122), and Gly(107)-Tyr(119), respectively. The measured mass difference values of the two labels agreed with that expected for CH3 /CD3, 3.019 Ha, with a standard deviation of 0.005 Da, providing persu asive identity verification. MS/MS fragmentation narrowed the site to pro(109)-Phe(118) and also caused loss of the derivative with a sulfur atom, uniquely identifying Cys(113) as the thiaminase I active-site n ucleophile among the 379 amino acids.