{
  "abstract": "Global quantification of DNA cytosine modifications, including 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC), is important for understanding cancer biology, though established methods require multistep workflows and costly instrumentation. Here, we show that attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy combined with regression modeling enables rapid, label-free, and nondestructive quantification of both modifications from DNA samples. Using Adenomatous Polyposis Coli (APC) promoter DNA standards spanning 0-100% modification, we identified modification-sensitive spectral features and observed that 5-hmC produces greater spectral changes than 5-mC. A univariate peak-ratio approach yielded strong linearity for both modifications (R ² = 0.97), while partial least-squares regression (PLSR) improved quantification accuracy to R ² = 0.99 (RMSE = 2.6%) for 5-hmC and R ² = 0.97 (RMSE = 5.7%) for 5-mC. In composite mixtures containing all three cytosine states, 5-hmC remained highly quantifiable (R ² = 0.97; RMSE = 5.1%), while 5-mC accuracy decreased (R ² = 0.90; RMSE = 9.6%), consistent with the greater spectral distinctiveness secondary to the hydroxymethyl group. Transferability was assessed using circulating tumor DNA (ctDNA), short cell-free DNA fragments shed from tumor cells into the bloodstream, comprising multiplexed reference material spanning seven genomic regions and a polydisperse fragment-length distribution (155-220 bp). After domain adaptation between synthetic and ctDNA spectra, we obtained a quantitative methylation calibration with R ² = 0.98 and RMSE = 5.2% under cross-validation. These results support ATR-FTIR spectroscopy as a viable platform for global cytosine modification quantification and establish proof-of-concept applicability to ctDNA analysis.",
  "abstract_shown": true,
  "affiliations": [
    "Optoelectronics Research Centre, Building 53, University of Southampton, University Road, Southampton, Hampshire SO17 1BJ, United Kingdom.",
    "The Breast Cancer Now Toby Robins Research Centre, The Institute of Cancer Research, Mary-Jean Mitchell Green Building, Chester Beatty Laboratories, 237 Fulham Road, London SW3 6JB, United Kingdom.",
    "The Royal Marsden NHS Foundation Trust, Fulham Road, London SW3 6JJ, United Kingdom."
  ],
  "article_type": "research article",
  "arxiv": null,
  "author_total": 3,
  "authors": [
    {
      "affs": [
        0
      ],
      "name": "Rashad Fatayer",
      "orcid": "0000-0002-6105-0760"
    },
    {
      "affs": [
        1,
        2
      ],
      "name": "Stephen-John Sammut",
      "orcid": "0000-0003-4472-904X"
    },
    {
      "affs": [
        0
      ],
      "name": "Ganapathy Senthil Murugan",
      "orcid": "0000-0002-2733-3273"
    }
  ],
  "bibtex": "https://research.draconitic.com/paper/3x7ybmht8nwhg.bib",
  "cited_by": 1,
  "doi": "10.1021/acsmeasuresciau.6c00167",
  "fulltext_url": "https://europepmc.org/articles/PMC13693256",
  "id": "3x7ybmht8nwhg",
  "issns": [
    "2694-250X"
  ],
  "landing_url": "https://europepmc.org/article/MED/42921427",
  "language": "eng",
  "licence": "CC-BY",
  "licence_label": "CC BY",
  "licence_source": "article",
  "licence_url": "https://creativecommons.org/licenses/by/4.0/",
  "metadata_licence": null,
  "native_subjects": [
    [
      "openalex",
      "1304|Spectroscopy Techniques in Biomedical and Chemical Research"
    ]
  ],
  "openalex": "W7171379751",
  "pdf_url": "https://europepmc.org/articles/PMC13693256?pdf=render",
  "pmcid": "PMC13693256",
  "pmid": "42921427",
  "published": "2026-07-24",
  "publisher": null,
  "ris": "https://research.draconitic.com/paper/3x7ybmht8nwhg.ris",
  "sources": [
    {
      "first_seen": "2026-10-30T12:21:02Z",
      "native_id": "MED:42921427",
      "source": "europepmc",
      "url": "https://europepmc.org/article/MED/42921427"
    }
  ],
  "subjects": [
    {
      "field": "biochemistry",
      "origin": "openalex:1304|Spectroscopy Techniques in Biomedical and Chemical Research",
      "subfield": "biophysics"
    }
  ],
  "title": "DNA Methylation and Hydroxymethylation Quantification Using Vibrational Spectroscopy.",
  "title_text": "DNA Methylation and Hydroxymethylation Quantification Using Vibrational Spectroscopy.",
  "type": "peer-reviewed",
  "updated_at": "2026-10-30T12:21:02Z",
  "url": "https://research.draconitic.com/paper/3x7ybmht8nwhg",
  "venue": "ACS measurement science au",
  "versions": []
}