Fast and High-Throughput Evaluation of Photodynamic Effect by Monitoring Specific Protein Oxidation with MALDI-TOF Mass Spectrometry

Abstract

In antibacterial practices by photodynamic treatment, bacteria are incubated with photosensitizers and then oxidized to death by generating reactive oxygen species (ROS) under light irradiation. Generally, Luria-Bertani (LB) agar colony is a conventional method to evaluate the photodynamic effect. However, this method is time consuming, easily disturbed by pollutants, and limited to the analysis of a pure bacteria sample. Herein, we introduce a novel method of photodynamic effect evaluation through in situ detection of specific protein oxidation by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) with only 1 μL of sample in a fast (less than 1 min per sample) and high-throughput (up to 384 samples per run) way. The oxidation rates of specific proteins stayed highly consistent with bactericidal rates and thus MALDI-TOF MS might be able to replace the LB agar colony to evaluate the photodynamic effect. With the present method, several experimental conditions including different photosensitizer types, dosage controls, and different illumination times were easily screened to optimize photodynamic effect. Photodynamic effects of various bacteria species, cancer cells, and even mixture samples were also evaluated. The results demonstrate the promising application of MALDI-TOF MS in evaluating the photodynamic effect of each component in a mixture sample without any separation or purification, which could not be achieved by the traditional LB agar colony method.