An analytical workflow for the molecular dissection of irreversibly modified fluorescent proteins

Abstract

Owing to their ability to be genetically expressed in live cells, fluorescent proteins have become indispensable markers in cellular and biochemical studies. These proteins can undergo a number of covalent chemical modifications that may affect their photophysical properties. Among other mechanisms, such covalent modifications may be induced by reactive oxygen species (ROS), as generated along a variety of biological pathways or through the action of ionizing radiations. In a previous report [1], we showed that the exposure of cyan fluorescent protein (ECFP) to amounts of (•)OH that mimic the conditions of intracellular oxidative bursts (associated with intense ROS production) leads to observable changes in its photophysical properties in the absence of any direct oxidation of the ECFP chromophore. In the present work, we analyzed the associated structural modifications of the protein in depth. Following the quantified production of (•)OH, we devised a complete analytical workflow based on chromatography and mass spectrometry that allowed us to fully characterize the oxidation events. While methionine, tyrosine, and phenylalanine were the only amino acids that were found to be oxidized, semi-quantitative assessment of their oxidation levels showed that the protein is preferentially oxidized at eight residue positions. To account for the preferred oxidation of a few, poorly accessible methionine residues, we propose a multi-step reaction pathway supported by data from pulsed radiolysis experiments. The described experimental workflow is widely generalizable to other fluorescent proteins, and opens the door to the identification of crucial covalent modifications that affect their photophysics.

Free Open Source Software for Protein and Peptide Mass Spectrometry- based Science

“…One most eloquent example is the study of tubulin poly-glutamylation or poly-glycylation, where the substrate is made of a large number of protein isoforms and the post-translational poly-modifications themselves are poly-disperse [62,63]. In a large spectrum of fields of endeavor, involving induced chemical modifications of proteins or peptides, human eye scrutiny of the obtained mass spectrometric data is crucial to extract data bits not predictably modelled by automated workflows [64,65]. Finally, one example of compulsory mass spectral data visualization is when isotopic cluster profiles need to be compared between experiments with differential isotopic labelling, like in pulse-chase experiments.…”