In humans, neutrophils represent the initial line of defense against bacterial pathogens. Invading bacteria are killed by neutrophils by a process called phagocytosis, during which bacteria are exposed to a cocktail of oxidative substances. One of these substances is the highly toxic hypochlorous acid (HOCl), which is generated by the Myeloperoxidase (MPO) in the phagolysosome. In proteins HOCl chiefly oxidizes thiols but also reacts with free amines to form N-chloramines, often resulting in a loss of protein function. However, there are also proteins that are turned into effective holdase-like chaperones by N-chlorination. Despite the clear effect of N-chlorination on protein function, there are currently no strategies to study the extent of N-chloramines in vivo.

We have recently shown that protein N-chlorination can be detected in vitro using the DNASyl derivative DANSyl sulfinic acid (DANSO₂H). Additionally free amines can be labeled by DANSyl chloride (DANSCl). Both reactions yield the same DANSylated fluorescent protein product. To advance insights into the role of N-chlorination in host-pathogen interactions, this study aims to harness this chemistry in combination with isotopic labeling to establish a robust, quantifiable labeling protocol to detect N-chloramines in vitro and in vivo.

Labeling of N-chloramines and free amines was initially optimized on peptides and proteins in vitro and followed up by the establishment of labeling procedures in vivo. The addition of detergents during the labeling process was found to significantly increase labeling efficiency. This increase in labeling suggests that not all amines/N-chloramines are accessible for DANSyl labeling without denaturation. The labeling degree was quantified through in-gel fluorescence measurements of the DANSyl products and confirmed by mass spectrometry analysis.

The DANSyl labeling established here lays a foundation for the quantification of the occurrence of N-chloramines in vivo and to study their pathophysiological role.