{
  "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.",
  "abstract_shown": true,
  "affiliations": [
    "Multi-Omics Mass Spectrometry Facility, Suzhou Institute of Systems Medicine, Chinese Academy of Medical Sciences & Peking Union Medical College, Suzhou, 215123"
  ],
  "article_type": "New results",
  "arxiv": null,
  "author_total": 7,
  "authors": [
    {
      "name": "X.-F. Yang"
    },
    {
      "name": "A.-C. Hou"
    },
    {
      "name": "Q. Sun"
    },
    {
      "name": "P.-Z. Mao"
    },
    {
      "name": "Y.-J. Qin"
    },
    {
      "name": "X.-M. Zhang"
    },
    {
      "affs": [
        0
      ],
      "name": "G.-C. Shao"
    }
  ],
  "bibtex": "https://research.draconitic.com/paper/5ntddg9bn4bv9.bib",
  "cited_by": 0,
  "doi": "10.64898/2026.09.02.748724",
  "fulltext_url": "https://www.biorxiv.org/content/10.64898/2026.09.02.748724v1.full",
  "id": "5ntddg9bn4bv9",
  "issns": [],
  "landing_url": "https://www.biorxiv.org/content/10.64898/2026.09.02.748724v1",
  "language": null,
  "licence": "CC-BY",
  "licence_label": "CC BY",
  "licence_source": "article",
  "licence_url": "https://creativecommons.org/licenses/by/4.0/",
  "metadata_licence": null,
  "native_subjects": [
    [
      "biorxiv",
      "biochemistry"
    ],
    [
      "openalex",
      "1607|Mass Spectrometry Techniques and Applications"
    ]
  ],
  "openalex": "W7207617236",
  "pdf_url": "https://www.biorxiv.org/content/10.64898/2026.09.02.748724v1.full.pdf",
  "pmcid": null,
  "pmid": null,
  "published": "2026-09-02",
  "publisher": null,
  "ris": "https://research.draconitic.com/paper/5ntddg9bn4bv9.ris",
  "sources": [
    {
      "first_seen": "2026-10-30T12:19:07Z",
      "native_id": "10.64898/2026.09.02.748724",
      "source": "biorxiv",
      "url": "https://www.biorxiv.org/content/10.64898/2026.09.02.748724v1"
    }
  ],
  "subjects": [
    {
      "field": "chemistry",
      "origin": "openalex:1607|Mass Spectrometry Techniques and Applications",
      "subfield": "physical"
    }
  ],
  "title": "Enhancing the Identification of NHS Ester-Mediated Lysine-Cysteine Cross-Linking via Reduced Trypsin Digestion Time",
  "title_text": "Enhancing the Identification of NHS Ester-Mediated Lysine-Cysteine Cross-Linking via Reduced Trypsin Digestion Time",
  "type": "preprint",
  "updated_at": "2026-10-30T12:19:07Z",
  "url": "https://research.draconitic.com/paper/5ntddg9bn4bv9",
  "venue": "bioRxiv",
  "versions": []
}