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One of the most widely used methods to detect an acute viral infection in clinical specimens is diagnostic real-time polymerase chain reaction. However, because of the COVID-19 pandemic, mass-spectrometry-based proteomics is currently being discussed as a potential diagnostic method for viral infections. Because proteomics is not yet applied in routine virus diagnostics, here we discuss its potential to detect viral infections. Apart from theoretical considerations, the current status and technical limitations are considered. Finally, the challenges that have to be overcome to establish proteomics in routine virus diagnostics are highlighted.

Parallel-reaction monitoring (PRM) using high resolution, accurate mass (HR/AM) analysis on quadrupole-Orbitrap mass spectrometers, like the Q Exactive, is one of the most promising approaches for targeted protein analysis. However, PRM has a limited multiplexing capacity, which depends heavily on the reproducibility of peptide retention times. To overcome these limitations, we aimed to establish an easily applicable data acquisition mode that allows retention-time-independent massive multiplexing on Q Exactive mass spectrometers. The presented method is based on data-dependent acquisition and is called pseudo-PRM. In principle, high-intensity stable isotope-labeled peptides are used to trigger the repeated fragmentation of the corresponding light peptides. In this way, pseudo-PRM data can be analyzed like normal PRM data. We tested pseudo-PRM for the target detection from yeast, human cells, and serum, showing good reproducibility and sensitivities comparable to normal PRM. We demonstrated further that pseudo-PRM can be used for accurate and precise quantification of target peptides, using both precursor and fragment ion areas. Moreover, we showed multiplexing of more than 1000 targets in a single run. Finally, we applied pseudo-PRM to quantify vaccinia virus proteins during infection, verifying that pseudo-PRM presents an alternative method for multiplexed target profiling on Q Exactive mass spectrometers.