Tunable electron activated dissociation (EAD) MS/MS to preserve particularly labile post-translational modifications
Site-localization of malonylated peptides using the SCIEX ZenoTOF 7600 system
Joanna Bons1, Jason Cason2, Birgit Schilling1, Christie Hunter3
1Buck Institute, USA, 2SCIEX, Canada, 3SCIEX, USA
Post-translational modifications (PTMs) are important players in a diverse group of functions that include protein conformation and signaling. Lysine acylation, such as malonylation, is one such PTM, and is regulated in part by lysine deacylases, which are members of the sirtuin (SIRT) protein family. In a previous study investigating SIRT5-regulated lysine malonylome, it was shown that 183 malonyllysine sites (from 120 proteins) out of the 1,137 identified sites (from 430 proteins) were significantly increased in Sirt5-/- KO versus wild-type mice.1 Specifically, it revealed that malonylation regulated GADPH activity. Malonylated peptides are however traditionally difficult to characterize using mass spectrometry and CID because the modification is extremely labile.
In this work, that involves using EAD technology with tunable kinetic energy, the effects of kinetic energy ramping on the preservation of highly labile PTMs (malonylation, for example) was studied, focusing on one previously identified malonyllysine site from GADPH (K-192). Two orthogonal fragmentation modes were compared (EAD vs. CID), to investigate the utility of each for PTM site localization. In addition, samples were measured using MRMHR mode to investigate the use of EAD for quantitative PTM characterization of labile modifications.2 MS/MS data were acquired with the Zeno trap activated, which provides significant sensitivity increases and enhances the quality of EAD MS/MS spectra.