A top-down approach to protein structure studies using chemical cross-linking and Fourier transform mass spectrometry.
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 cross-linkers 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 A), disuccinimidyl glutarate (DSG, cross-linker arm length = 7.5 A) and disuccinimidyl tartrate (DST, cross- linker arm length = 5.8 A) are homobifunctional cross-linking reagents that react specifically with primary amines. Using tandem mass spectrometry (MS/MS) on the singly, internally cross-linked 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 approximate, equals K6 approximate, equals K48 approximate, equals 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.
Novak
Petr
P
Sandia National Laboratories, PO Box 969, Livermore, CA 94551-0969, USA.
Young
Malin M
MM
Schoeniger
Joseph S
JS
Kruppa
Gary H
GH
D000595:Amino Acid Sequence; D003432:Cross-Linking Reagents / Q000737:chemistry*; D005583:Fourier Analysis; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D013329:Structure-Activity Relationship; D025801:Ubiquitin / Q000737:chemistry
D003432:Cross-Linking Reagents; D011506:Proteins; D025801:Ubiquitin