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.