Abstract
The challenge of discovering a completely new human tumor virus of unknown phylogeny or sequence depends on detecting viral molecules and differentiating them from host molecules in the virus-associated neoplasm. We developed differential peptide subtraction (DPS) using differential mass spectrometry (dMS) followed by targeted analysis to facilitate this discovery. We validated this approach by analyzing Merkel cell carcinoma (MCC), an aggressive human neoplasm, in which ~80% of cases are caused by the human Merkel cell polyomavirus (MCV). Approximately 20% of MCC have a high mutational burden and are negative for MCV, but are microscopically indistinguishable from virus positive cases. Using 23 (12 MCV+, 11 MCV–) formalin-fixed MCC, DPS identified both viral and human biomarkers (MCV large T antigen, CDKN2AIP, SERPINB5, and TRIM29) that discriminate MCV+ and MCV– MCC. Statistical analysis of 498,131 dMS features not matching the human proteome by DPS revealed 562 (0.11%) to be upregulated in virus-infected samples. Remarkably, 4 (20%) of the top 20 candidate MS spectra originated from MCV T oncoprotein peptides and confirmed by reverse translation degenerate oligonucleotide sequencing. DPS is a robust proteomic approach to identify potentially novel viral sequences in infectious tumors when nucleic acid–based methods are not feasible.
Highlights
Seven human viruses are responsible for approximately 15% of the tumor burden world-wide
We developed a methodology called differential peptide subtraction (DPS) using label-free differential mass spectrometry to quantify relative peptide abundance between complex samples [19,20,21]
Unbiased DPS was performed on polyomavirus-positive and -negative Merkel cell carcinoma (MCC)
Summary
Seven human viruses are responsible for approximately 15% of the tumor burden world-wide. This phylogenetically heterogeneous group of viruses differ extensively in their genome sizes, nucleic acid composition, and replication mechanisms [1]. The discovery processes for each of these 7 tumor viruses has varied and evolved closely with technological advances, in molecular biology. 2), a large double stranded DNA herpesvirus, was first identified in 1964 based on classic microbiology detection practices in cell culture and electron microscopy. Hepatitis B virus (HBV), unculturable at the time, was found by serologic screening in 1965 [3]. Hepatitis C virus (HCV), a flavivirus, was found by cDNA library screening in 1989 [6]
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