Ion coalescence of neutron encoded TMT 10-plex reporter ions

Isobaric mass tag-based quantitative proteomics strategies such as iTRAQ and TMT utilize reporter ions in the low mass range of tandem MS spectra for relative quantification. The recent extension of TMT multiplexing to 10 conditions has been enabled by utilizing neutron encoded tags with reporter ion m/z differences of 6 mDa. The baseline resolution of these closely spaced tags is possible due to the high resolving power of current day mass spectrometers. In this work we evaluated the performance of the TMT10 isobaric mass tags on the Q Exactive Orbitrap mass spectrometers for the first time and demonstrated comparable quantification accuracy and precision to what can be achieved on the Orbitrap Elite mass spectrometers. However, we discovered, upon analysis of complex proteomics samples on the Q Exactive Orbitrap mass spectrometers, that the proximate TMT10 reporter ion pairs become prone to coalescence. The fusion of the different reporter ion signals into a single measurable entity has a detrimental effect on peptide and protein quantification. We established that the main reason for coalescence is the commonly accepted maximum ion target for MS2 spectra of 1e6 on the Q Exactive instruments. The coalescence artifact was completely removed by lowering the maximum ion target for MS2 spectra from 1e6 to 2e5 without any losses in identification depth or quantification quality of proteins.