Large-scale Targeted Analysis of Post-Translational Modifications by Internal Standard-Triggered Parallel Reaction Monitoring: Application to Determination of Phosphorylation Site Occupancy

摘要

Targeted quantitative proteomic analyses performed on high-resolution and accuratemass (HRAM) spectrometers showed unique analytical performance for clinical samples. In such applications, parallel reaction monitoring (PRM) was carried out on quadrupole-orbitrap instruments, which showed significant increase in selectivity and sensitivity of measurements. The recent implementation of the internal standard triggered-PRM (IS-PRM, [1]) mode has enabled large-scale targeted experiments. Here, the field of application of the IS-PRM technique has been expanded to include the analysis of post-translational modifications. The approach was applied to the determination of phosphorylation site occupancy, which is crucial to gain a detailed understanding of the regulation mechanisms of cellular signaling pathways. In addition, the use of IS-PRM was explored in combination with fast liquid chromatography separations to increase analytical throughput.