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High-resolution human papillomavirus genotyping by MALDI-TOF mass spectrometry

Nature Protocols volume 3, pages 1476–1484 (2008)Cite this article

Abstract

We describe a matrix-assisted laser desorption/ionization–time of flight (MALDI-TOF) mass spectrometry (MS)-based assay for human papillomavirus (HPV) genotyping—the restriction fragment mass polymorphism (RFMP) assay, which is based on mass measurement of genotype-specific oligonucleotide fragments generated by TypeIIS restriction endonuclease cleavage after recognition sites have been introduced by PCR amplification. The use of a TypeIIS restriction enzyme makes the RFMP assay independent of sequence and applicable to a wide variety of HPV genotypes, because these enzymes have cleavage sites at a fixed distance from their recognition sites. After PCR amplification, samples are subjected to restriction enzyme digestion with FokI and BtsCI and desalting using Oasis purification plates, followed by analysis by MALDI-TOF MS. Overall, the protocol is simple, takes ∼4–4.5 h and can accurately detect and identify at least 74 different HPV genotypes.

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Figure 1: General procedures for the restriction fragment mass polymorphism genotyping assay.
Figure 2: Matrix-assisted laser desorption/ionization mass spectra of the restriction fragment mass polymorphism assays of three high-risk type human papillomaviruses (HPVs) in negative-ion mode.
Figure 3: Matrix-assisted laser desorption/ionization mass spectra of the restriction fragment mass polymorphism assays of three nonhigh-risk type human papillomaviruses (HPVs) in negative-ion mode.
Figure 4: Matrix-assisted laser desorption/ionization mass spectra of the restriction fragment mass polymorphism assays of mixed genotype infections in negative-ion mode.
Figure 5: Rescue experiment for a case with a failure in PCR amplification step.

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Authors and Affiliations

  1. R&D Center, GeneMatrix Inc, Yongin, 446-913, Korea

    Sun Pyo Hong, Soo-Kyung Shin, Eun Ok Kim, Seung Il Ji, Hyun Jae Chung, Wangdon Yoo & Soo-Ok Kim

  2. Department of Laboratory Medicine, Green Cross Reference Laboratory, Yongin, 446-913, Korea

    Eun Hee Lee

  3. Department of Toxicological Research, Korea Food and Drug Administration, National Institute of Toxicological Research, Seoul, 122-704, Korea

    Sun Nie Park

  4. Department of Biochemistry, University of Missouri–Columbia, Columbia, 65201-5149, Missouri, USA

    William R Folk

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Hong, S., Shin, SK., Lee, E. et al. High-resolution human papillomavirus genotyping by MALDI-TOF mass spectrometry. Nat Protoc 3, 1476–1484 (2008). https://doi.org/10.1038/nprot.2008.136

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