Poster Presentations
Day 2, June 11（Thu.） Room P (5F 501+502)
- 2P-17
Generation of TMT Complementary Ions by EAD for Quantitative Proteomics
(1Kyoto Univ., 2AB SCIEX)
oNishida Hiroshi1, Takeshi Shibata2, Ryo Yokoyama2, Kosuke Ogata1, Ushio Takeda2, Yasushi Ishihama1
Tandem Mass Tag (TMT)-based isobaric labeling enables multiplexed quantitative proteomics and is widely used for simultaneous analysis of multiple samples. However, conventional TMT quantification relies on low-mass reporter ions, which are susceptible to signal interference caused by co-fragmentation of co-isolated peptide ions, resulting in reduced quantitative accuracy. Complementary ion-based quantification (TMTc), which utilizes fragment ions in the high-mass region of MS/MS spectra, has been proposed as an alternative strategy to reduce such interference because these ions retain precursor mass information. TMT complementary ions are generated when the reporter group dissociates while the balancer part remains attached to the peptide. However, their generation efficiency depends strongly on fragmentation conditions such as collision energy and precursor charge state. In this study, we systematically investigated fragmentation conditions that enhance TMTc generation. Proteins extracted from HeLa cells were digested with trypsin and labeled with TMTpro reagents. Selected doubly and triply charged peptide ions were analyzed on a ZenoTOF 8600 system using CID and EAD under various collision energy conditions. CID generated strong 9plex TMTc ions, whereas EAD produced complementary ions retaining the balancer structure, enabling detection of 10-plex TMTc signals. These results suggest that EAD fragmentation seems to enable efficient generation of 10plex TMTc ions for complementary ion–based quantitative proteomics.