Triggered offset fragmentation allows massively multiplexed target profiling on quadrupole-orbitrap mass spectrometers

Parallel reaction monitoring (PRM) on quadrupole-orbitrap mass spectrometers has a limited multiplexing capacity which depends heavily on the reproducibility of peptide retention times. To overcome these limitations, we aimed to establish a data acquisition mode that allows retention-time-independent massive multiplexing on quadrupole-orbitrap 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 with Skyline. We tested pseudo-PRM for the target detection from yeast, human cells, and serum, and assessed the quantification accuracy/precision and sensitivity. Moreover, we analyzed multiplexing of more than 1,000 targets in a single pseudo-PRM 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 quadrupole-orbitrap mass spectrometers.