The detection of HPV infection and microbial colonization in cervical lesions is currently done through PCR-based viral or bacterial DNA amplification. Our objective was to develop a methodology to expand the metaproteomic landscape of cervical disease and determine if protein biomarkers from both human and microbes could be detected in distinct cervical samples. This would lead to the development of multi-species proteomics, which includes protein-based lateral flow diagnostics that can define patterns of microbes and/or human proteins relevant to disease status. In this study, we collected both non-frozen tissue biopsy and exfoliative non-fixed cytology samples to assess the consistency of detecting human proteomic signatures between the cytology and biopsy samples. Our results show that proteomics using biopsies or cytologies can detect both human and microbial organisms. Across patients, Lumican and Galectin-1 were most highly expressed human proteins in the tissue biopsy, whilst IL-36 and IL-1RA were most highly expressed human proteins in the cytology. We also used mass spectrometry to assess microbial proteomes known to reside based on prior 16S rRNA gene signatures. Lactobacillus spp. was the most highly expressed proteome in patient samples and specific abundant Lactobacillus proteins were identified. These methodological approaches can be used in future metaproteomic clinical studies to interrogate the vaginal human and microbiome structure and metabolic diversity in cytologies or biopsies from the same patients who have pre-invasive cervical intraepithelial neoplasia, invasive cervical cancer, as well as in healthy controls to assess how human and pathogenic proteins may correlate with disease presence and severity.

Aberdeen Centre for Women's Health Research University of Aberdeen Scotland UK

Aberdeen Royal Infirmary Foresterhill Road Aberdeen UK

Biochemistry and Microbiology University of Victoria Canada

Department of Pharmacology and Public Health Faculty of Medicine The Hashemite University Zarqa 13133 Jordan

Institute of Environmental Medicine Karolinska Institutet Stockholm Sweden

International Centre for Cancer Vaccine Science University of Gdansk Gdansk Poland

Research Centre for Applied Molecular Oncology Masaryk Memorial Cancer Institute 656 53 Brno Czech Republic

Scottish HPV Reference Laboratory Royal Infirmary of Edinburgh NHS Lothian UK UK

The Academy of Applied Medical and Social Sciences Lotnicza 2 Elblag Poland; Department of Medical Laboratory Diagnostics Fahrenheit Biobank BBMRI pl Medical University of Gdańsk Marii Skłodowskiej Curie 3a 80 210 Gdańsk Poland

The Academy of Applied Medical and Social Sciences Lotnicza 2 Elblag Poland; Gastrointestinal Surgical Oncology Department Greater Poland Cancer Centre Garbary 15 Poznan Poland

Unit of Gynaecologic Oncology 2nd Department of Obstetrics and Gynecology Aretaieion Hospital Medical School National and Kapodistrian University of Athens Greece

University of Edinburgh Institute of Genetics and Molecular Medicine Edinburgh Scotland UK

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