期刊
EUROPEAN JOURNAL OF MASS SPECTROMETRY
卷 9, 期 6, 页码 623-631出版社
SAGE PUBLICATIONS LTD
DOI: 10.1255/ejms.590
关键词
top-down; Fourier transform mass spectrometry; chemical cross-linking; proteins
in a preliminary communication we described a top-down approach to the determination of chemical cross-link location in proteins using Fourier transform mass spectrometry (FT-MS). We have since extended the approach to use a series of homobifunctional crosslinkers with the same reactive functional groups, but different cross-linker arm lengths. Correlating cross-linking data across a series of related linkers allows the distance constraint derived from a cross-link between two reactive side chains to be determined more accurately and increases the confidence in the assignment of the cross-links. In ubiquitin, there are seven lysines with primary amino groups and the amino terminus. Disuccinimidyl suberate (DSS, cross-linker arm length = 11.4 Angstrom), disuccinimidyl glutarate (DSG, cross-linker arm length = 7.5 Angstrom) and disuccinimidyl tartrate (DST, cross-linker arm length = 5.8 Angstrom) are homobifunctional cross-linking reagents that react specifically with primary amines. Using tandem mass spectrometry (MS/MS) on the singly, internally crosslinked precursor ion of ubiquitin, we found cross-links with DSS and DSG between the amino terminus and Lys 6, between Lys 6 and Lys 11, and between Lys 63 and Lys 48. Using disuccinimidyl tartrate (DST), the shortest cross-linker in the series, only the crosslinks between the amino terminus and Lys 6, and between Lys 6 and Lys 11 were observed. The observed cross-links are consistent with the crystal structure of ubiquitin, if the lysine side chains and the amino terminus are assumed to have considerable flexibility. In a separate study, we probed the reactivity of the primary amino groups in ubiquitin using the amino acetylating reagent, N-hydroxy succinimidyl acetate (NHSAc), and a top-down approach to localize the acetylated lysine residues. The reactivity order obtained in that study (M1approximate toKapproximate toK48approximate toK63) > K33 > K11 > (K27, K29), shows that the cross-link first formed in ubiquitin by reaction with DSS and DSG occurs between the most reactive residues.
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