Investigating Acquisition Performance on the Orbitrap Fusion When Using Tandem MS/MS/MS Scanning with Isobaric Tags

Methods for isobaric-tagged peptide analysis (e.g., TMT, such as the synchronous precursor selection (SPS) tandem MS/MS/MS (MS3) approach, enable maintenance of reporter ion accuracy and precision by reducing the ratio compression caused by coisolated precursor ions. However, the decreased throughput of the MS3 approach necessitates careful optimization of acquisition strategies and methods to ensure maximal proteome coverage. We present a systematic analysis of acquisition parameters used to analyze isobaric-tagged peptide samples on current generation Orbitrap mass spectrometer (MS) hardware. In contrast with previously reported works, we demonstrate the limited utility of acquiring reporter ion,data in the ion trap analyzer; ion trap acquisition had only a minimal increase in identification depth and reduced quantification precision. We establish that despite the significantly increased scan rate afforded through the use of higher energy collisional dissociation (HCD) in MS3-based ion trap isobaric tag analyses, the reduced quantification precision and reporter ion yields negate the potential benefits in proteome coverage. Lastly, using optimized parameter sets, we further demonstrate the limited utility of the ion trap detector versus the Orbitrap for reporter ion detection in an in-depth analysis of a complex proteome sample. Together, these data will serve as a valuable resource to researchers undertaking analysis on current generation Orbitrap instrumentation with isobaric tags.