期刊
JOURNAL OF MASS SPECTROMETRY
卷 39, 期 3, 页码 322-328出版社
WILEY
DOI: 10.1002/jms.587
关键词
top-down; Fourier transform mass spectrometry; solvent accessibility; proteins
We present a method employing top-down Fourier transform mass spectrometry (FTMS) for the rapid profiling of amino acid side-chain reactivity. The reactivity of side-chain groups can be used to infer residue-specific solvent accessibility and can also be used in the same way as H/D exchange reactions to probe protein structure and interactions. We probed the reactivity of the N-terminal and epsilon-lysine amino groups of ubiquitin by reaction with N-hydroxysuccinimidyl acetate (NHSAc), which specifically acetylates primary amines. Using a hybrid Q-FTMS instrument, we observed several series of multiply acetylated ubiquitin ions that varied with the NHSAc: protein stoichiometry. We isolated and fragmented each member of the series of acetylated ubiquitin ions in the front end of the instrument and measured the fragment ion masses in the FTMS analyzer cell to determine which residue positions were modified. As we increased the NHSAc: protein stoichiometric ratio, identification of the fragments from native protein and protein with successively increasing modification allowed the assignment of the complete order of reactivity of the primary amino groups in ubiquitin (Met 1 approximate to Lys 6 approximate to Lys 48 approximate to Lys 63 > Lys 33 > Lys 11 > Lys 27, Lys 29). These results are in excellent agreement with the reactivity expected from other studies and predicted from the known crystal structure of ubiquitin. The top-down approach eliminates the need for proteolytic digestion, high-performance liquid chromatographic separations and all other chemical steps except the labeling reaction, making it rapid and amenable to automation using small quantities of protein. Copyright (C) 2004 John Wiley Sons, Ltd.
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