Mass Spectrometry-Based Proteomics of Minor Species in the Bulk: Questions to Raise with Respect to the Untargeted Analysis of Viral Proteins in Human Tissue.

(1) Background: Untargeted mass spectrometry (MS)-based proteomic analysis is highly amenable to automation. Software algorithms translate raw spectral data into protein information obtained by a comparison to sequence databases. However, the technology has limitations, especially for analytes measured at the limit of detection. In a protein expression study of human gastric biopsies, the question arose whether or not it is possible, as well as sensible, to search for viral proteins in addition to those from the human host. (2) Methods: Experimental data-independent MS data were analyzed using protein sequences for oncoviruses, and BLAST analyses were performed to elucidate the level of sequence homology to host proteins. (3) Results: About one hundred viral proteins were assigned, but there was also up to 43% sequence homology to human proteins. (4) Conclusions: There are at least two reasons why the matches to viral proteins should be used with care. First, it is not plausible that large amounts of viral proteins should be present in human gastric biopsies, so the spectral quality of the peptides derived from viral proteins is likely low. As a consequence, the number of false assignments is high. Second, homologous peptides found both in human and virus proteomes contribute to matching errors. Thus, though shotgun proteomics raw data can technically be analyzed using any database, meaningful results cannot be always expected and a sanity check must be performed. Both instrumentation and bioinformatic processing in MS-based proteomics are continuously improving at lowering the limit of detection even further. Nevertheless, data output should always be controlled in order to avoid the over-interpretation of results.