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Titolo:
IDENTIFICATION OF MODIFICATION SITES IN LARGE BIOMOLECULES BY STABLE-ISOTOPE LABELING AND TANDEM HIGH-RESOLUTION MASS-SPECTROMETRY - THE ACTIVE-SITE NUCLEOPHILE OF THIAMINASE-I
Autore:
KELLEHER NL; NICEWONGER RB; BEGLEY TP; MCLAFFERTY FW;
Indirizzi:
CORNELL UNIV,BAKER LAB,DEPT CHEM ITHACA NY 14853 CORNELL UNIV,BAKER LAB,DEPT CHEM ITHACA NY 14853
Titolo Testata:
The Journal of biological chemistry
fascicolo: 51, volume: 272, anno: 1997,
pagine: 32215 - 32220
SICI:
0021-9258(1997)272:51<32215:IOMSIL>2.0.ZU;2-O
Fonte:
ISI
Lingua:
ENG
Soggetto:
INFRARED MULTIPHOTON DISSOCIATION; MULTIPLY-CHARGED IONS; ELECTROSPRAY; PROTEINS; FRAGMENTS; RESONANCE; ANALOGS; PHASE;
Tipo documento:
Article
Natura:
Periodico
Citazioni:
41
Recensione:
Indirizzi per estratti:
Citazione:
N.L. Kelleher et al., "IDENTIFICATION OF MODIFICATION SITES IN LARGE BIOMOLECULES BY STABLE-ISOTOPE LABELING AND TANDEM HIGH-RESOLUTION MASS-SPECTROMETRY - THE ACTIVE-SITE NUCLEOPHILE OF THIAMINASE-I", The Journal of biological chemistry, 272(51), 1997, pp. 32215-32220

Abstract

A widely used procedure for site localization of covalent protein modifications involves proteolysis, partial chromatographic separation ofthe resulting complex mixture, and tandem mass spectrometry (MS/MS) to identify peptides whose molecular weight (M-r) has been increased appropriately by the modification, As found previously for MS of small molecules, this study shows that protein fragment identification can begreatly 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 70peptides, FTMS identification of the labeled peptide localized the modification site of a mechanism-based inhibitor to Arg(101)-Lys(121), Asp(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 persuasive identity verification. MS/MS fragmentation narrowed the site topro(109)-Phe(118) and also caused loss of the derivative with a sulfur atom, uniquely identifying Cys(113) as the thiaminase I active-site nucleophile among the 379 amino acids.

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Documento generato il 05/07/20 alle ore 21:57:27