Enhancing the Identification of NHS Ester-Mediated Lysine-Cysteine Cross-Linking via Reduced Trypsin Digestion Time
- 1Multi-Omics Mass Spectrometry Facility, Suzhou Institute of Systems Medicine, Chinese Academy of Medical Sciences & Peking Union Medical College, Suzhou, 215123
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 due to the extreme lability of the resulting thioester bonds. Here, we demonstrate that NHS esters efficiently label cysteine residues and form stable lysine-cysteine (K-C) cross-links--species previously deemed labile in XL-MS. To preserve these thioester-based cross-linked sites , we optimized the standard XL-MS workflow and applied it to four model proteins. K-C cross-links constitute 23% to 59% of total detected cross-links, with C-C distances predominantly ranging from 12 to 40 [A], and 60% satisfying theoretical linker constraints -- confirming that K-C cross-links are as reliable as canonical lysine-lysine (K-K) cross-link sites. Cross-software validation and benchmarking against the heterobifunctional K-C cross-linker GMBS further demonstrated that our optimized workflow captures ~ 40% of GMBS-identified K-C cross-linked sites. Collectively, these findings expand the practical utility of NHS esters and enhance protein structural characterization by enabling simultaneous acquisition of K-K and K-C distance constraints.
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BibTeX
@misc{yang2026enhancing,
title = {{Enhancing the Identification of NHS Ester-Mediated Lysine-Cysteine Cross-Linking via Reduced Trypsin Digestion Time}},
author = {X.-F. Yang and A.-C. Hou and Q. Sun and P.-Z. Mao and Y.-J. Qin and X.-M. Zhang and G.-C. Shao},
howpublished = {bioRxiv},
year = {2026},
month = sep,
doi = {10.64898/2026.09.02.748724},
url = {https://www.biorxiv.org/content/10.64898/2026.09.02.748724v1},
note = {CC BY},
}
RIS
TY - UNPB
TI - Enhancing the Identification of NHS Ester-Mediated Lysine-Cysteine Cross-Linking via Reduced Trypsin Digestion Time
AU - X.-F. Yang
AU - A.-C. Hou
AU - Q. Sun
AU - P.-Z. Mao
AU - Y.-J. Qin
AU - X.-M. Zhang
AU - G.-C. Shao
PY - 2026
DA - 2026/09/02
PB - bioRxiv
DO - 10.64898/2026.09.02.748724
UR - https://www.biorxiv.org/content/10.64898/2026.09.02.748724v1
AB - 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 due to the extreme lability of the resulting thioester bonds. Here, we demonstrate that NHS esters efficiently label cysteine residues and form stable lysine-cysteine (K-C) cross-links--species previously deemed labile in XL-MS. To preserve these thioester-based cross-linked sites , we optimized the standard XL-MS workflow and applied it to four model proteins. K-C cross-links constitute 23% to 59% of total detected cross-links, with C-C distances predominantly ranging from 12 to 40 [A], and 60% satisfying theoretical linker constraints -- confirming that K-C cross-links are as reliable as canonical lysine-lysine (K-K) cross-link sites. Cross-software validation and benchmarking against the heterobifunctional K-C cross-linker GMBS further demonstrated that our optimized workflow captures ~ 40% of GMBS-identified K-C cross-linked sites. Collectively, these findings expand the practical utility of NHS esters and enhance protein structural characterization by enabling simultaneous acquisition of K-K and K-C distance constraints.
ER -