Directly mapping the O-sulfation sites within CCR2 via an engineered nanopore.

Abstract

Tyrosine O-sulfation is one of the significant post-translational modifications that participates in and mediates various life activities in cells. However, until now, the detailed functions of tyrosine O-sulfation have hardly been understood. Although mass spectrometry, ELISA, and radioactive isotope-label have been explored to study tyrosine O-sulfation, the in-depth characterization of this post-translational modification is still rare due to the lack of general, label-free methods for accurately mapping and quantifying the tyrosine O-sulfation sites. Specifically, for mass spectrometry, sulfotyrosine is easily broken down at high temperatures and low pH, usually leading to the loss of sulfated modification in both positive and negative ion MS/MS signals. Moreover, since sulfotyrosine and phosphotyrosine have a similar mass change around 80 Da (sulfotyrosine: 79.9568 Da; phosphotyrosine: 79.9663 Da), it is quite difficult to directly discriminate them unless using ultra-high accuracy mass measurements. Therefore, mass spectrometry still faces huge limitations in rapid sulfotyrosine identification and quantification. Recently, nanopore has raised its potential for single protein sequencing and achieved the detection of various post-translational modifications, such as phosphorylation, glycosylation, methylation, etc. Here, mutant aerolysin nanopores are used for the detection of sulfotyrosine and the mapping of its site without labeling. It should be noted that the experiments are performed under mild conditions without impact on the stability of sulfotyrosine. The results show that phosphorylated, sulfated and nonmodified peptides can be clearly distinguished by differences in the relative current blockades. Furthermore, the mapping of the sites of sulfated tyrosine on CCR2 was achieved by further improving the sensitivity of aerolysin nanopore. These findings offer a path to the single-molecule, label-free identification of sulfotyrosine.