{
  "abstract": "In bottom-up proteomics, protease specificity determines peptide length and LC/MS/MS detectability; in N-terminomics, this is critical because each protein N-terminal peptide is unique and cannot be substituted by other peptides. CHromatographic AMplification of Protein N-terminal peptides (CHAMP-N) combines LysargiNase digestion with StageTip-based strong cation exchange chromatography (SCX), removing internal peptides by exploiting their newly generated N-terminal Lys/Arg residues and thereby selectively isolating protein N-terminal peptides. However, LysargiNase cleavage near the protein N-terminus can generate excessively short peptides. Here, we developed two complementary CHAMP-N approaches that modurate N-terminal peptide length while retaining the charge-based SCX separation principle. LysN digestion avoided cleavage at N-terminal-proximal Arg residues, whereas D3-acetylation blocked Lys cleavage by LysargiNase to produce ArgN-like specificity. Both approaches extended otherwise short N-terminal peptides while preserving N-terminal basic residues on internal peptides. Conventional LysargiNase, LysN, and ArgN-like CHAMP-N generated complementary sets of N-terminal peptides. Integration of the three workflows identified 2,552 protein N-termini, approximately twice the number identified by conventional CHAMP-N alone. The median relative standard deviations of N-terminal peptide intensities were approximately 15% for all three workflows, indicating comparable technical reproducibility. These results demonstrate that coordinating protease cleavage specificity with SCX selectivity reduces sequence-dependent losses and expands N-terminome coverage while preserving the simplicity and reproducibility of CHAMP-N.",
  "abstract_shown": true,
  "affiliations": [
    "Kyoto University"
  ],
  "article_type": "New results",
  "arxiv": null,
  "author_total": 4,
  "authors": [
    {
      "name": "A. Takeshita"
    },
    {
      "name": "E. Kanao"
    },
    {
      "name": "K. Imami"
    },
    {
      "affs": [
        0
      ],
      "name": "Y. Ishihama"
    }
  ],
  "bibtex": "https://research.draconitic.com/paper/4zcn7813rc1w7.bib",
  "cited_by": 0,
  "doi": "10.64898/2026.09.22.753420",
  "fulltext_url": "https://www.biorxiv.org/content/10.64898/2026.09.22.753420v1.full",
  "id": "4zcn7813rc1w7",
  "issns": [],
  "landing_url": "https://www.biorxiv.org/content/10.64898/2026.09.22.753420v1",
  "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|Advanced Proteomics Techniques and Applications"
    ]
  ],
  "openalex": "W7214087876",
  "pdf_url": "https://www.biorxiv.org/content/10.64898/2026.09.22.753420v1.full.pdf",
  "pmcid": null,
  "pmid": null,
  "published": "2026-09-22",
  "publisher": null,
  "ris": "https://research.draconitic.com/paper/4zcn7813rc1w7.ris",
  "sources": [
    {
      "first_seen": "2026-10-30T12:24:00Z",
      "native_id": "10.64898/2026.09.22.753420",
      "source": "biorxiv",
      "url": "https://www.biorxiv.org/content/10.64898/2026.09.22.753420v1"
    }
  ],
  "subjects": [
    {
      "field": "chemistry",
      "origin": "openalex:1607|Advanced Proteomics Techniques and Applications",
      "subfield": "physical"
    }
  ],
  "title": "Expanding N-terminomics Coverage by Extending N-terminal Peptide Length",
  "title_text": "Expanding N-terminomics Coverage by Extending N-terminal Peptide Length",
  "type": "preprint",
  "updated_at": "2026-10-30T12:24:00Z",
  "url": "https://research.draconitic.com/paper/4zcn7813rc1w7",
  "venue": "bioRxiv",
  "versions": []
}