Stable Isotope-Triggered Offset Fragmentation Allows Massively Multiplexed Target Profiling on Quadrupole-Orbitrap Mass Spectrometers.

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.