Improved 6-Plex Tandem Mass Tags Quantification Throughput Using a Linear Ion Trap–High-Energy Collision Induced Dissociation MS^3 Scan

The use of tandem mass tags (TMT) as an isobaric labeling strategy is a powerful method for quantitative proteomics, yet its accuracy has traditionally suffered from interference. This interference can be largely overcome by selecting MS^2 fragment precursor ions for high-energy collision induced dissociation (HCD) MS^3 analysis in an Orbitrap scan. While this approach minimizes the interference effect, sensitivity suffers due to the high AGC targets and long acquisition times associated with MS^3 Orbitrap detection. We investigated whether acquiring the MS^3 scan in a linear ion trap with its lower AGC target would increase overall quantification levels with a minimal effect on precision and accuracy. Trypsin-digested proteins from Saccharomyces cerevisiae were tagged with 6-plex TMT reagents. The sample was subjected to replicate analyses using either the Orbitrap or the linear ion trap for the HCD MS^3 scan. HCD MS^3 detection in the linear ion trap vs Orbitrap increased protein identification by 66% with minor loss in precision and accuracy. Thus, the use of a linear ion trap–HCD MS^3 scan during a 6-plex TMT experiment can improve overall identification levels while maintaining the power of multiplexed quantitative analysis.