Developing new isotope-coded mass spectrometry-cleavable cross-linkers for elucidating protein structures.
Structural characterization of protein complexes is essential for the understanding of their function and regulation. However, it remains challenging due to limitations in existing tools. With recent technological improvements, cross-linking mass spectrometry (XL-MS) has become a powerful strategy to define protein-protein interactions and elucidate structural topologies of protein complexes. To further advance XL-MS studies, we present here the development of new isotope-coded MS-cleavable homobifunctional cross-linkers: d0- and d10-labeled dimethyl disuccinimidyl sulfoxide (DMDSSO). Detailed characterization of DMDSSO cross-linked peptides further demonstrates that sulfoxide-containing MS-cleavable cross-linkers offer robust and predictable MS2 fragmentation of cross-linked peptides, permitting subsequent MS3 analysis for simplified, unambiguous identification. Concurrent usage of these reagents provides a characteristic doublet pattern of DMDSSO cross-linked peptides, thus aiding in the confidence of cross-link identification by MS(n) analysis. More importantly, the unique isotopic profile permits quantitative analysis of cross-linked peptides and therefore expands the capability of XL-MS strategies to analyze both static and dynamic protein interactions. Together, our work has established a new XL-MS workflow for future studies toward the understanding of structural dynamics of protein complexes.
Department of Physiology and Biophysics, University of California , Medical Science I, D233, Irvine, California 92697, United States.
Clinton
C
Yu
Wynne
W
Kandur
Athit
A
Kao
Scott
S
Rychnovsky
Lan
L
Huang
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D003432:Cross-Linking Reagents / Q000737:chemistry*; D007554:Isotopes; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics
D003432:Cross-Linking Reagents; D007554:Isotopes; D010446:Peptide Fragments