BPS2026 – Lysine succinylation in charge rich proteins measured using a novel label free tool

Succinylation is a post-translational modification that is essential for regulating several cellular and molecular processes and is implicated in multiple diseases. This modification alters the positive charge of the lysine, making it negative. Conventionally, PTMs have been detected using antibody-based techniques and spectroscopic methods, such as protein labeling with external probes, NMR, mass spectrometry (MS), FTIR, and western blotting. However, these conventional techniques require expensive instrumentation and specific antibodies, increasing the overall cost. Currently, there are limited methods available that can detect PTMs without labeling, using low-cost instruments, and in a quick time. Here, we introduce a novel intrinsic chromophore in proteins that exhibits photo-induced electron transfer transitions. This photo-induced charge transfer arises from charged amino acids and the polypeptide backbone. It produces a broad UV-visible absorption band ranging from 250 to 800 nm. This newly identified absorbance band is called Protein Charge-Transfer Spectra (ProCharTS). It offers a new label-free approach for probing protein post-translational modifications, as it depends on the charge content of amino acids in proteins. Succinylation alters the charge; thus, we hypothesize it will significantly reflect in the ProCharTS. To validate our hypothesis, we explore the detection of chemical succinylation in charge-rich proteins α 3 C, α 3 W and Histone H2A using ProCharTS. Initially, we confirmed protein succinylation and its different extents through MALDI-ToF and analyzed the secondary structure by CD spectroscopy. The ProCharTS spectra of succinylated proteins were recorded, demonstrating a decrease in intensity for α 3 C, α 3 W and histone H2A. ProCharTS was sensitive to detect single lysine succinylation in proteins. It exhibits a significant decrease across different degrees of succinylation. Our results showed that the reduction in the ProCharTS intensity after succinylation is due to the loss of positive charge in the proteins.