Joanna Bons Joanna Bons ,Ph.D., is a postdoctoral fellow in the laboratory of Dr. Birgit Schilling at the Buck Institute for Research on Aging. After an engineer degree in Biotechnology, she joined the team of Dr. Christine Carapito at the BioOrganic Mass Spectrometry Laboratory in Strasbourg, France, where she specialized in quantitative mass spectrometry-based proteomics method development (SRM, PRM, DIA) for proteome quantification and characterization. She received her Ph.D. in Analytical Chemistry in 2019, and then joined Dr. Birgit Schilling’s laboratory. She focuses on developing and optimizing innovative DIA and targeted strategies, using CID/HCD and electron activated dissociation (EAD), for deciphering proteome and PTM remodeling in various collaborative projects.

ZenoTOF 7600 Acquisitions with Electron Activated Dissociation and Novel Skyline Features for Quantification of Protein Post-translational Modifications

Protein post-translational modifications (PTMs) are key players involved in many cellular processes and signaling. Proteomic analysis of PTMs however can be challenging, due to the presence of labile modifications, the low stoichiometry of PTMs, the presence of multiple PTMs per peptide, and modified isomeric peptides. Collision induced dissociation (CID) has been commonly used for PTM analysis, but the relatively high fragmentation energy applying CID fragmentation can lead to side chain losses and incomplete sequence coverage for some modified peptides. An alternative and milder fragmentation mechanism, electron activated dissociation (EAD), implemented in the novel ZenoTOF 7600 Q-TOF system (SCIEX), was very recently introduced, and generates z+1-ion and c-ion series. We assessed the performances of EAD fragmentation to preserve PTM groups, and improve labile PTM characterization, site localization and quantification, and compared with CID fragmentation. Read More
Specifically, a series of synthetic post-translationally modified peptides, featuring phosphorylated, succinylated, malonylated and acetylated mono- and doubly-modified isomeric peptides, were analyzed using targeted high-resolution multiple reaction monitoring (MRM-HR) assays, with or without Zeno trap activation. EAD tunable kinetic energy values were ramped from 0 eV to 11 eV, and optimized for each analyte, conferring efficient EAD product ion fragmentation, namely z+1-ion and c-ion series. Acquired EAD MRM-HR data was analyzed using Skyline to extract peak areas for c, z+1 and b and y fragment ion series, as well as their counterparts with neutral loss. Indeed, taking advantage of the novel z+1 fragment ion support implemented into Skyline, we were able to benefit from the user-friendly Skyline features for EAD data visualization and quantification.Finally, this novel MS workflow, combining EAD MRM-HR and Skyline data processing, to a cohort of Sirt5(-/-) and WT human K562 cells enriched for malonyl peptides in order to gain insights into the regulation of Sirtuin 5, a NAD+-dependent lysine deacylase, enzymatically removing malonyl groups.


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