Evaluating Multiplexed Quantitative PhosphopeptideAnalysis on a Hybrid Quadrupole Mass Filter/Linear Ion Trap/OrbitrapMass Spectrometer

摘要

As a driver for many biological processes, phosphorylation remains an area of intense research interest. Advances in multiplexed quantitation utilizing isobaric tags (e.g., TMT and iTRAQ) have the potential to create a new paradigm in quantitative proteomics. New instrumentation and software are propelling these multiplexed workflows forward, which results in more accurate, sensitive, and reproducible quantitation across tens of thousands of phosphopeptides. This study assesses the performance of multiplexed quantitative phosphoproteomics on the Orbitrap Fusion mass spectrometer. Utilizing a two-phosphoproteome model of precursor ion interference, we assessed the accuracy of phosphopeptide quantitation across a variety of experimental approaches. These methods included the use of synchronous precursor selection (SPS) to enhance TMT reporter ion intensity and accuracy. We found that (i) ratio distortion remained a problem for phosphopeptide analysis in multiplexed quantitative workflows, (ii) ratio distortion can be overcome by the use of an SPS-MS3 scan, (iii) interfering ions generally possessed a differentcharge state than the target precursor, and (iv) selecting only thephosphate neutral loss peak (single notch) for the MS3 scan stillprovided accurate ratio measurements. Remarkably, these data suggestthat the underlying cause of interference may not be due to coelutingand cofragmented peptides but instead from consistent, low level backgroundfragmentation. Finally, as a proof-of-concept 10-plex experiment,we compared phosphopeptide levels from five murine brains to fivelivers. In total, the SPS-MS3 method quantified 38 247 phosphopeptides,corresponding to 11 000 phosphorylation sites. With 10 measurementsrecorded for each phosphopeptide, this equates to more than 628 000binary comparisons collected in less than 48 h.