Enhancing the Identification of NHS Ester-Mediated Lysine-Cysteine Cross-Linking via Reduced Trypsin Digestion Time
Abstract Chemical cross-linking mass spectrometry (XL-MS) is a powerful technique for elucidating protein structures and interactions, with NHS ester-based cross-linkers being the most widely used. Traditionally, NHS esters are considered highly specific for primary amines; although their reactivity toward cysteine thiols has been reported, it has not been systematically characterized in XL-MS…
A study has revealed that NHS ester-mediated lysine-cysteine (K-C) cross-linking can be reliably identified in chemical cross-linking mass spectrometry (XL-MS) despite previous concerns about the stability of these bonds. By optimizing the XL-MS workflow, researchers have demonstrated that K-C cross-links, which were once considered labile, constitute 23% to 59% of total detected cross-links in four model proteins.
These cross-links have C-C distances ranging from 12 to 40 angstroms, and 60% of them meet theoretical linker constraints, proving their reliability comparable to canonical lysine-lysine (K-K) cross-link sites. Furthermore, software validation and comparison with another heterobifunctional K-C cross-linker, GMBS, showed that the optimized workflow captures approximately 40% of GMBS-identified K-C cross-linked sites.
This advancement significantly expands the practical use of NHS esters and facilitates simultaneous acquisition of K-K and K-C distance constraints, thereby enhancing protein structural characterization.
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