A Top-Down Approach to Protein Structure Studies Using Chemical Cross-Linking and Fourier Transform Mass Spectrometry
- 1 December 2003
- journal article
- Published by SAGE Publications in European Journal of Mass Spectrometry
- Vol. 9 (6), 623-631
- https://doi.org/10.1255/ejms.590
Abstract
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 Å), disuccinimidyl glutarate (DSG, cross-linker arm length = 7.5 Å) and disuccinimidyl tartrate (DST, cross-linker arm length = 5.8 Å) 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 cross-links 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 (M1 ≈ K6 ≈ K48 ≈ K63) > 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.Keywords
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