Detection of HPV at the Protein Level

ThinPrep smear testing is the standard method of screening cervical cells for abnormalities and presence of the human papillomavirus (HPV), a known tumor-associated virus. The risk of cervical cancer as a result of HPV infection varies based on the particular strain, as some strains pose a higher risk than others. Smear test screenings allow cancerous and precancerous cells to be identified for early therapeutic intervention.¹

To date, the cervical smear remains largely unexplored by proteomics; however, some protein characterizations have been successful using 2-DIGE² or LC-MS on laser-captured, microdissected ThinPrep slides.³ High-resolution proteomic analysis of HPV could identify HPV biomarkers leading to better assessment of cancer risk level and developing cancer, as well as a greater understanding of the HPV integration process at the protein level.

In their study,⁴ the Garbis group performed a proteomic analysis of females positive for HPV and females with no cervical dysplasia. A total of 23 cervical smear specimens were obtained for analysis, which included 17 high- and/or low-risk HPV-positive females and six HPV-negative females. Positive samples were selected to include as many different strains of HPV as possible.

Proteins were purified, digested, and labeled with iTRAQ. The peptides were then fractionated using high-pH reverse-phase fractionation. LC-MS was performed on three 8-plex sample sets using a LTQ-Velos Orbitrap-Elite mass spectrometer (Thermo Scientific) coupled to a Dionex Ultimate 3000 UHPLC system. Nitrogen high-collision dissociation (HCD MS/MS) and helium collision-induced dissociation (CID MS/MS) peptide fragmentation modes were used.

Mass spectra were submitted to the Sequest search engine implemented on the Proteome Discoverer software version 1.3.0.339. A strict false-discovery rate of <1% was established using the Percolator node along with database searching in a UniProt Fasta file containing 20,200 human reviewed entries and a total of 7,180 human HPV sequences.

This analysis yielded the identification of 17,580 unique peptides corresponding to 3,217 unique proteins, and over 2,310 proteins were quantitatively profiled in at least one of the three experiments. The Garbis group suggests that state-of-the-art 2D LC-FT-Orbitrap-based techniques alongside traditional methods will increase the sensitivity and specificity of current screening approaches.

References

1. Whitlock, E.P., et al. (2011) ‘Liquid-based cytology and human papillomavirus testing to screen for cervical cancer: a systematic review for the U.S. Preventive Services Task Force‘, Annals of Internal Medicine, 155 (10), (pp. 687–697)

2. Rader, J.S., et al. (2008) ‘A unified sample preparation protocol for proteomic and genomic profiling of cervical swabs to identify biomarkers for cervical cancer screening‘, Proteomics. Clinical Applications, 2 (12), (pp. 1658–1669)

3. Gu, Y., et al. (2007) ‘Proteomic analysis of high-grade dysplastic cervical cells obtained from ThinPrep slides using laser capture microdissection and mass spectrometry‘, Journal of Proteome Research, 6 (11), (pp. 4256–4268)

4. Papachristou, E.K., et al. (2013) ‘The shotgun proteomic study of the human ThinPrep cervical smear using iTRAQ Mass-Tagging and 2D LC-FT-Orbitrap-MS: The detection of the human papillomavirus at the protein level‘, Journal of Proteome Research, March 19, 2013, doi: /10.1021/pr301067r